Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 1 Epigenetic regulation of skeletal development of fetuses from HFD (high fat diet) rat dams. A, Representative images from Alizarin red/Alcian blue staining of E18.5 embryos from dams fed either control diet or HFD. Cartilage and calcification stained dark blue; arrows indicate differences of skeletal ossification in the head. B, Western blots for H3K27me3, Ezh2, p-Ezh2 and CDK1 in proteins from EOCCs (embryonic rat osteogenic calvarial cells) either from six HFD obese dams or six control diet dams. M, male; F, female. C, Real-time PCR for Ezh2 mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. D, and E, representing top genes and significantly H3K27me3 enriched or decreased gene body and gene promoter using Heat Map analysis of ChIP-seq data from EOCCs either from six HFD obese dams or six control diet dams (pooled)

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, ChIP-sequencing

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 2 Identification of epigenetic target genes affected by maternal obesity during fetal skeletal development. A, SATB2 was significantly enriched for H3K27me3 within the gene body, especially in the promoter region based on the detection of a peak (black and red arrow head) of enrichment by Illumina DNA sequencing (male and female are mixed, and samples were pooled to three per group). B, Real-time PCR for SATB2 and ALP mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. C, ChIP of rat SATB2 enhancer elements by specific anti H3K27me3 and Ezh2 antibodies. D, ChIP of enrichment of SATB2 after IP with H3K27me3 and Ezh2 antibodies. Fold enrichment relative to IgG. *P < .05 by t-test EOCCs from control vs EOCCs from HFD obese dams. E, Control EOCCs were treated with vehicle (control), NEFA mixture 400 µM with Palmitic and Oleic acid 2:1 mixture, Sigma-Aldrich), Palmitic acid (270 µM) or Oleic acid (130 µM) for 48 hours: Western blots for H3K27me3, Ezh2, p-Ezh2, SATB2 and Col 1 (collagen 1). n = 3/treatment

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 4 Increased Ezh2/H3K27me3 but decreased SATB2 expression in human UC MSCs from obese mothers. A, UC MSCs from either lean or obese mothers were cultured, passage 2 cells were immune-stained with anti-Ezh2 antibody (red, white arrows) or anti-SATB2 antibody (green, yellow arrows). B, and C, Real-time PCR (Box & Whiskers graphs) and Western blots (under Box & Whiskers graphs) of Ezh2 and SATB2 mRNA and protein expression in UC MSCs either from lean or obese mothers. *P < .05 vs lean, t-test. D, ChIP of human Ezh2, SATB2 and GAPDH enhancer elements by specific anti H3K27me3 antibody, and E, ChIP of enrichment of human Ezh2, SATB2 and GAPDH after IP with H3K27me3 antibody, fold enrichment relative to IgG. *P < .01 by t-test vs lean

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 6 Increased trabecular bone mineral density in Ezh2 osteoblastic cell specific deletion male mice. A, Sagittal views of total, trabecular and cortical bone mineral density, and Strength Strain Index (SSI) from a represented mouse from cko and their respective control mice. B, Total bone mineral content in all female mice. B, Tibia pQCT parameters, TOT-BMC (total bone mineral content), P = .0499 by one-way ANOVA; TOT-BMD (total bone mineral density), P = .0365 by one-way ANOVA; TRAB-BMD (trabecular bone mineral density), P = .0188 by one-way ANOVA; CRT-BMD (cortical bone mineral density), P = .0620 by one-way ANOVA in male cko mice compared to their respective genotypic control mice, followed by Tukey's post hoc test comparing cko with its respective control genotype group, *means P < .05 significantly different, n = 4

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Control

Journal: Oncogene

Article Title: RASSF10 is frequently epigenetically inactivated in kidney cancer and its knockout promotes neoplasia in cancer prone mice

doi: 10.1038/s41388-020-1195-6

Figure Lengend Snippet: a RASSF10 expression across human normal tissues (log2, data GTEX— n = 2921—RPKM—ensgtexv4). b Kidney cancer cell lines were analyzed by combined bisulfite restriction analysis (+ Taq I digested; −mock digest) for RASSF10 promoter hypermethylation and 9 out of 15 (60%) were methylated (m). HeLa was used as positive control. HEK293 cell line is partially methylated (pm). c Pharmacological inhibition of DNA-methyl-transferases by 5-Aza-2′deoxycytidine (Aza) significantly reestablished RASSF10 expression in kidney cancer cell lines MZ1257 and MZ1973 ( t -test). RASSF10 was normalized to ß-ACTIN . d In kidney cancer patient samples RASSF10 methylation (cg05817758 in beta value) blocks RASSF10 expression (log2; norm. rsem + 1) shown for renal clear cell and e renal papillary carcinoma. Analyzed using . f RASSF10 inhibition by CRISPR-Cas9 genomic targeting of RASSF10. HEK293 (RASSF10-TetOn) cells were transfected with CRISPR-Cas9 guide RNAs in px549 vector targeting RASSF10 (three guide combinations) for 48 h and RASSF10 was induced by doxycycline for 24 h. g RASSF10 is epigenetically regulated by EZH2 and p300. HEK293 (RASSF10-TetOn) cells were transfected with RASSF10 guide RNAs in px549 (deadCas) targeting RASSF10 and recruiting EZH2(deadCas) or p300(deadCas) for 72 h and RASSF10 was induced by doxycycline for 24 h. Protein lysates were separated by SDS-Page and western blotted with indicated antibodies.

Article Snippet: EZH2 (Ezh2[SET]-dCas9 Addgene) and p300 (pcDNA-dCas9-p300 Core Addgene) were used as epigenetic modifiers upon cotransfection in HEK293 with guide RNAs(dCas).

Techniques: Expressing, Methylation, Positive Control, Inhibition, CRISPR, Transfection, Plasmid Preparation, SDS Page, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 6. Physical interaction between DUXAP9 and EZH2 in OSCC cells. A) DUXAP9-interacting proteins were separated by SDS-PAGE followed by an endogenous DUXAP9 RNA pull-down assay and manifested by Coomassie Brilliant Blue staining. The distinct protein bands in the gel were excised,

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: SDS Page, Pull Down Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 7. DUXAP9 increases EZH2 protein expression via inhibition of its proteasomal degradation. A) The mRNA expression of EZH2 was measured by qRT-PCR in CAL27 and HN6 cells transfected with SS-NC or SS-DUXAP9 ASO (left) or control- or DUXAP9-overexpressing vectors (right). B) The

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Transfection, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 8. DUXAP9 suppresses EZH2 degradation via inhibition of the phosphorylation (Thr345/Thr487) of EZH2. A,B) Western blot showing the levels of p-EZH2 (T345), p-EZH2 (T487), and CDK1 in DUXAP9-silenced A) and DUXAP9-overexpressing B) CAL27 and HN6 cells treated with MG132 (20 μm

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 9. DUXAP9 suppresses EZH2 degradation via nuclear-to-cytoplasmic translocation. A–C) Western blot showing the levels of nuclear and cy- toplasmic EZH2 in CDK1- A) and DUXAP9- B) overexpressing and DUXAP9 knockdown C) CAL27 and HN6 cells. Quantification of the EZH2 nu- cleus/cytoplasm ratio by grayscale analysis is shown on the right. D) Western blot shows the levels of nuclear and cytoplasmic mutant EZH2 in vector- or CDK1-overexpressing 293T cells. E) Western blot shows the levels of nuclear and cytoplasmic wild-type EZH2 and mutant EZH2 in CDK1-overexpressing 293T cells. F) Western blot shows the levels of nuclear and cytoplasmic EZH2 in 293T cells transfected with control or mutant DUXAP9-overexpressing vectors in combination with CDK1-overexpressing vector. Data are presented as the mean ± SD from three independent experiments. Data in (A–F) were calculated by two-tailed unpaired Student’s t-test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Translocation Assay, Western Blot, Knockdown, Mutagenesis, Plasmid Preparation, Transfection, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 10. DUXAP9 promotes the proliferation and invasion of OSCC cells by mediating EZH2 expression and function. A) The mRNA expression of 9 known EZH2 target genes was analyzed by qRT-PCR assays in CAL27 and HN6 cells transfected with vector, DUXAP9, and EZH2. B) ChIP-qPCR assay of EZH2 or IgG occupancy at the CDKN1A, DAB2IP, and RUNX2 loci in CAL27 cells transfected with SS-NC or SS-DUXAP9. C) The volumes, weights and growth curves of tumors derived from CAL27 cells transfected with control- or DUXAP9-expressing vectors and siRNAs targeting NC or EZH2 were measured and imaged; D,E) The expression of Ki67, PCNA, and EZH2 was determined by the IHC score in the indicated groups. n = 5/group, Scale bars, 100 μm (left), 25 μm (right). Data in (C) are presented as the mean ± SEM, and data in (A,B) and (D) are presented as the mean ± SD from three independent experiments. Data were calculated by two-tailed unpaired Student’s t-test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, ChIP-qPCR, Derivative Assay, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Figure 11. Schematic depicting YY1-induced DUXAP9 drives OSCC by blocking CDK1-mediated EZH2 degradation. DUXAP9 orchestrates a different biological function of CDK1-mediated phosphorylation of the T345 and T487 sites of EZH2 in controlling the protein stability of EZH2 via nuclear to cytoplasmic translocation, suggesting the importance of lncRNA regulation at the posttranslational level in OSCC progression.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Blocking Assay, Phospho-proteomics, Translocation Assay

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: EZH2 expression in 3 endometrial cancer cell lines (ECC-1, HEC1-A, RL95-2) and the normal endometrium cell line T-HESC. (A) Real time PCR (B) Western Blot (C) ECC-1, HEC1-A and RL95-2 cell lines were transfected with shEZH2 or control (scEZH2). Drug selected cells were examined for expression of EZH2 and β-actin by western blotting. As indicated, stable knock down clones were created and isolated for all cell lines. All experiments were completed in triplicate with representative images shown.

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Knockdown, Clone Assay, Isolation

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: EZH2 knockdown inhibited cell proliferation, migration and invasion. (A) Equal number of control and shEZH2 knock down cells were assayed for proliferation via MTT assay. A significant decrease in proliferation was noted. (B, C) Control and shEZH2 cell lines were assayed for migration through control and Matrigel™ coated membranes. Cells that had migrated through the membrane were stained with 1% crystal violet in PBS and counted. All experiments were completed in triplicate

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Knockdown, Migration, Control, MTT Assay, Membrane, Staining

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: EZH2 silencing induced G2/M phase accumulation. Cell cycle assays illustrate an increase in the G2/M fraction in shEZH2 HEC1-A, ECC-1 and RL95-2 cell lines relative to control (❖ = p < 0.05). The image is representative of 3 separate experiments.

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Control

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: EZH2 silencing via RNA interference induces the expression of SFRP1, DKK3 and E-cadherin, and decreases β-catenin expression. A) Protein levels of the Wnt inhibitors SFRP1 and DKK3, down stream β-catenin as well as E-Cadehrin are shown in both control and EZH2 knockdown cell lines. β-Actin was used as a loading control in these experiments. B) Real time PCR expression of the above markers is also shown. A representative blot was shown from 3 independent experiments.

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, Control, Knockdown, Real-time Polymerase Chain Reaction

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: Correlation between EZH2 expression and clinico-pathologic variables

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing

Journal: International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Article Title: Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines

doi: 10.1097/IGC.0b013e318296a265

Figure Lengend Snippet: Expression of EZH2 in human endometrial cancer tissue samples. (A and B) Strong glandular and stromal staining. (C and D) Weak glandular and stromal staining. (Magnification × 400)

Article Snippet: EZH2 short hairpin (sh)RNA stable transfection Four individual 29mer EZH2 shRNA constructs, as well as a scrambled negative control non-effective shRNA (scEZH2), packaged in a retroviral green fluorescent protein (GFP) vector were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, Staining

Journal: Acta pharmacologica Sinica

Article Title: EZH2 regulates expression of FOXC1 by mediating H3K27me3 in breast cancers.

doi: 10.1038/s41401-020-00543-x

Figure Lengend Snippet: Fig. 4 Expression of FOXC1 was reversibly related to histone methylation (H3K27me3) by EZH2. a In silico analysis of the Oncomine datasets showed that FOXC1 expression was inversely related to H3K27me3 expression. b The expression of FOXC1 was inversely related to the expression of EZH2. c Schematic of FOXC1 regulation. EZH2 and EZH1 methylate H3K27 whereas KDM6A and KDM6B demethylate H3K27me. d Expression of FOXC1, EZH2, and H3K27me3 in different breast cancer cell lines. The experiments were performed in triplicate.

Article Snippet: Cells were then transfected with EZH2 expression plasmids (OriGene, Rockville, MD, USA) or an empty vector (OriGene, Rockville, MD, USA) using LipofectamineTM 3000 transfection reagent (Invitrogen, Grand Island, NY, USA) for 24 h. The cells were then incubated with 600 μg/mL geneticin for 3 weeks.

Techniques: Expressing, Methylation, In Silico

Journal: Acta pharmacologica Sinica

Article Title: EZH2 regulates expression of FOXC1 by mediating H3K27me3 in breast cancers.

doi: 10.1038/s41401-020-00543-x

Figure Lengend Snippet: Fig. 5 Overexpression of EZH2 inhibited the expression of FOXC1 by increasing H3K27me3 level. a Overexpression of EZH2 resulted in the methylation of histone H3 and the formation of H3K27me3, which inhibited the expression of FOXC1. b The knockdown of EZH2 expression reduced the levels of H3K27me3 and increased the expression of FOXC1 in MCF-7 cells. c The knockdown of EZH2 expression reduced the expression of H3K27me3 and increased the expression of FOXC1 in T47D cells. d Neither H3K27me3 nor FOXC1 expression was changed in HCC1806 EZH1 cells overexpressing EZH1 compared to control cells. e Neither H3K27me3 nor FOXC1 expression was changed in HCC1806 EZH1 shRNA cells, in which EZH1 was downregulated compared to control cells. The experiments were performed in triplicate. f In silico analysis from ENCODE showed that the H3K27me3 antibody can bind to the promoter region of the FOXC1 gene. g ChIP with H3K27me3 antibodies or control IgG showed that H3K27me3 antibodies bound to the promoter of the FOXC1 gene.

Article Snippet: Cells were then transfected with EZH2 expression plasmids (OriGene, Rockville, MD, USA) or an empty vector (OriGene, Rockville, MD, USA) using LipofectamineTM 3000 transfection reagent (Invitrogen, Grand Island, NY, USA) for 24 h. The cells were then incubated with 600 μg/mL geneticin for 3 weeks.

Techniques: Over Expression, Expressing, Methylation, Knockdown, Control, shRNA, In Silico

Journal: Acta pharmacologica Sinica

Article Title: EZH2 regulates expression of FOXC1 by mediating H3K27me3 in breast cancers.

doi: 10.1038/s41401-020-00543-x

Figure Lengend Snippet: Fig. 6 Inhibition of EZH2 in MCF-7 cells induced the expression of FOXC1. a–c Western blot analysis showed that the expression of FOXC1 was significantly increased by treatment of DZNeP in MCF-7 cells. d–f The expression of FOXC1 was significantly increased by treatment of GSK343 in MCF-7 cells. The experiments were performed in triplicate.

Article Snippet: Cells were then transfected with EZH2 expression plasmids (OriGene, Rockville, MD, USA) or an empty vector (OriGene, Rockville, MD, USA) using LipofectamineTM 3000 transfection reagent (Invitrogen, Grand Island, NY, USA) for 24 h. The cells were then incubated with 600 μg/mL geneticin for 3 weeks.

Techniques: Inhibition, Expressing, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: EZH2 regulates expression of FOXC1 by mediating H3K27me3 in breast cancers.

doi: 10.1038/s41401-020-00543-x

Figure Lengend Snippet: Fig. 7 Inhibition of demethylases KDM6A and KDM6B did not alter the expression of FOXC1 in breast cancer cells. a Expression of FOXC1, KDM6A, KDM6B, EZH2, and EZH1 in MCF-7, T47D, and HCC1806 cells. b Treatment with GSKJ1 did not change the expression of FOXC1 in HCC1806 cells. c Treatment of GSKJ1 did not change the expression of FOXC1 in BT549 cells.

Article Snippet: Cells were then transfected with EZH2 expression plasmids (OriGene, Rockville, MD, USA) or an empty vector (OriGene, Rockville, MD, USA) using LipofectamineTM 3000 transfection reagent (Invitrogen, Grand Island, NY, USA) for 24 h. The cells were then incubated with 600 μg/mL geneticin for 3 weeks.

Techniques: Inhibition, Expressing

Journal: Acta pharmacologica Sinica

Article Title: EZH2 regulates expression of FOXC1 by mediating H3K27me3 in breast cancers.

doi: 10.1038/s41401-020-00543-x

Figure Lengend Snippet: Fig. 9 Proposed mechanism: EZH2 regulates the expression of FOXC1 by mediating H3K27me3 in breast cancers. FOXC1 is highly expressed in TNBC. The expression of FOXC1 is epigenetically regulated by H3K27me3 and associated with doxorubicin resistance and a poor prognosis in patients with TNBC.

Article Snippet: Cells were then transfected with EZH2 expression plasmids (OriGene, Rockville, MD, USA) or an empty vector (OriGene, Rockville, MD, USA) using LipofectamineTM 3000 transfection reagent (Invitrogen, Grand Island, NY, USA) for 24 h. The cells were then incubated with 600 μg/mL geneticin for 3 weeks.

Techniques: Expressing

Journal: PloS one

Article Title: Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM.

doi: 10.1371/journal.pone.0033729

Figure Lengend Snippet: Figure 1. Loss of the let-7 family inversely correlated with increased expression of EZH2 in PCa tissue specimens compared to adjacent normal prostate tissues. (A) Microarray profiling was done for assessing the expression of miRNA using total RNA extracted from three PCa tissue specimens and matched adjacent normal prostate tissues. The results showed that let-7a, let-7b, let-7c and let-7d was highly expressed in prostate tissues and their expression was lost in human PCa tissue specimens (* p,0.05, ** p,0.01). (B) The results from real time-RT-PCR confirmed that the levels of let-7b, let-7c and let-7d were significantly down-regulated in tumors with higher Gleason grade. (7a: let-7a; N: normal; TG6: tumor tissues from patients with Gleason grade 6. n = 39 for normal, n = 44 for TG6, n = 52 for TG7, n = 33 for TG8, 9). (C) A significant up-regulation of the expression of EZH2 was observed in PCa tissue specimens with Gleason grade 7 (n = 46) and higher (n = 33), but not in PCa specimens with Gleason grade 6 (n = 39). (D) EZH2 levels were inversely correlated with let-7b and let-7c expressions in tumor specimens with Gleason grade 7. doi:10.1371/journal.pone.0033729.g001

Article Snippet: Luciferase activity assay PC3 PDGF-D cells with lower levels of let-7 were seeded at a density of 66103 cells/well in a 96-well plate and incubated for 24 h. The cells were co-transfected with EZH2 39UTR luciferase plasmid (Origene, Rockville, MD) or Renilla luciferase plasmid and control miRNA, let-7a, let-7b, let-7c, and let-7d precursors using DharmaFECT duo transfection reagent (DHARMACON, Lafayette, CO).

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: PloS one

Article Title: Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM.

doi: 10.1371/journal.pone.0033729

Figure Lengend Snippet: Figure 2. Let-7 regulated EZH2 expression, and inhibited clonogenic growth capacity of PCa cell lines. (A) Expression of EZH2 was found to be higher in PCa cell lines compared with immortalized prostate epithelial cell lines: PZ-HPV-7 and RWPE-1 (upper panel) and transfection of let-7 precursors inhibited EZH2 expression in PC3 and PC3 PDGF-D cells 3 days after transfection (middle and lower panel). (B) let-7 family members repressed EZH2 39UTR luciferase activity in PC3 PDGF-D cells (lower levels of let-7 family in these cells) co-transfected with let-7 and EZH2 39UTR luciferase plasmid. (C) let-7 binding sites in the 39UTR of EZH2 mRNA were shown. (D) Transfection of let-7 precursors significantly reduced clonogenic growth capacity of PC3 PDGF-D cells (Con: control, 7a: pre-let-7a, ** p,0.01). doi:10.1371/journal.pone.0033729.g002

Article Snippet: Luciferase activity assay PC3 PDGF-D cells with lower levels of let-7 were seeded at a density of 66103 cells/well in a 96-well plate and incubated for 24 h. The cells were co-transfected with EZH2 39UTR luciferase plasmid (Origene, Rockville, MD) or Renilla luciferase plasmid and control miRNA, let-7a, let-7b, let-7c, and let-7d precursors using DharmaFECT duo transfection reagent (DHARMACON, Lafayette, CO).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Binding Assay, Control

Journal: PloS one

Article Title: Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM.

doi: 10.1371/journal.pone.0033729

Figure Lengend Snippet: Figure 3. BR-DIM treatment increased let-7 and consequently reduced EZH2 expression. (A) Total RNA was isolated from LNCaP, C4-2B and PC3 cells treated with 25 mM BR-DIM for 24 h and the results from real time RT-PCR showing that the expression of let-7 was increased following BR-DIM treatment compared to untreated control (c: DMSO control). (B) Levels of EZH2 mRNA were repressed by BR-DIM treatment in a dose dependent meaner. (C and D) The cell lysates were prepared from cells treated with BR-DIM for 48 h and Western blot showing the protein levels of EZH2, which was down-regulated by BR-DIM treatment (PC3 PD: PC3 PDGF-D cells, *, p,0.05; **, p,0.01). doi:10.1371/journal.pone.0033729.g003

Article Snippet: Luciferase activity assay PC3 PDGF-D cells with lower levels of let-7 were seeded at a density of 66103 cells/well in a 96-well plate and incubated for 24 h. The cells were co-transfected with EZH2 39UTR luciferase plasmid (Origene, Rockville, MD) or Renilla luciferase plasmid and control miRNA, let-7a, let-7b, let-7c, and let-7d precursors using DharmaFECT duo transfection reagent (DHARMACON, Lafayette, CO).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Western Blot

Journal: PloS one

Article Title: Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM.

doi: 10.1371/journal.pone.0033729

Figure Lengend Snippet: Figure 4. BR-DIM intervention in PCa patients resulted in the increased expression of let-7 family and consequently inhibited EZH2 expression in tumor tissues. RNA was obtained from BR-DIM intervention clinical trial samples where BR-DIM was given to patients for 2–4 weeks prior to surgery. RNA was also obtained from formalin-fixed paraffin-embedded (FFPE) tissue specimens of PCa patients with matched tumor Gleason grade, tumor stage and patient age as control group. The expression of miRNAs and mRNA was assessed using real time RT-PCR. Relative miRNA and mRNA levels were normalized to RNU1A1 and beta-actin, respectively. (A) BR-DIM intervention led to the increased trend in levels of Let-7a expression. (B and C) let-7b, let-7c and let-7d were significantly up-regulated by BR-DIM intervention. (D) EZH2 expression was down-regulated by BR- DIM intervention. doi:10.1371/journal.pone.0033729.g004

Article Snippet: Luciferase activity assay PC3 PDGF-D cells with lower levels of let-7 were seeded at a density of 66103 cells/well in a 96-well plate and incubated for 24 h. The cells were co-transfected with EZH2 39UTR luciferase plasmid (Origene, Rockville, MD) or Renilla luciferase plasmid and control miRNA, let-7a, let-7b, let-7c, and let-7d precursors using DharmaFECT duo transfection reagent (DHARMACON, Lafayette, CO).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Control, Quantitative RT-PCR