Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. Expression of ERG, ESE1 and ESE3 determined by qRT-PCR in normal prostate and prostate tumors. Tumors are grouped according to the predominantly expressed ETS factor. B. Principal component analysis. Dots represent individual samples with their location determined by the principal components of the transcriptome. C. Number of differentially expressed genes with Q≤0.1 in each ETS subgroup. D-E. Venn diagrams showing shared and distinct differentially expressed genes among the indicated tumor subgroups.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A . Number of unique, similar and common features among the differentially expressed genes compared to normal prostate in ERG high , ESE3 low , ESE1 high and NoETS tumors according to Metacore. B. Commonly affected GeneGo Pathway Maps in the tumor subgroups. C. Differentially affected GeneGo Pathway Maps in ERG high , ESE3 low , ESE1 high and NoETS tumors.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques:

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. EZH2 level in normal prostate, ESE3 low and NoETS tumors. B. EZH2 level in NoETS and ESE3 low tumors in Wallace et al. microarray dataset. C. EZH2 level in control (sh-) and stable ESE3 knock-down (sh 4, 6, 7) LNCaP clones determined by RT-PCR. D. EZH2 level in control (sh-) and ESE3 knock-down (sh17, 28) LHS clones determined by RT-PCR. E. Nkx3.1 level in normal prostate, ESE3 low and NoETS tumors. F. Nkx3.1 level in control (sh-) and ESE3-knock-down LNCaP and LHS cells determined by RT-PCR and Western blot ( right bottom ). G. Nkx3.1 promoter activity in PC3 cells transfected with human NKx3.1 promoter reporter along with the indicated expression vectors. Luciferase reporter activity was measured after 24 h. *p<0.01; **, p<0.001.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Microarray, Control, Knockdown, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Expressing, Luciferase

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. RNA was extracted from control (sh-) and ESE3 knock-down (sh7) LNCaP cells 48 h post-transfection with siEZH2 and control siRNA and analyzed by RT-PCR. B. H3K27 methylation was assessed in sh- and sh7 LNCaP cells by ChIP and qPCR with primers encompassing the EBS and ARE in the Nkx3.1 gene. ETS2 was used as negative control C. ESE3 binding to the EZH2 and Nkx3.1 promoter was assessed by ChIP and qPCR in sh- and sh7 LNCaP cells. ETS2 was used as negative control . D. ESE3 binding to the EZH2 and Nkx3.1 promoter in control and ERG-expressing (ERG-18) LNCaP cells assessed by ChIP and qPCR. ETS2 was used as negative control . *, p<0.01.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Control, Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Methylation, Negative Control, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. ERG over-expression and ESE3 knock-down (B) enhance cell migration. Cells were grown until confluence and starved for 24 h when a scratch was performed on the monolayer. Pictures were taken at 0 and 72 h. Representative photographs of triplicate experiments are shown. C. Control and ERG over-expressing LNCaP cell clones were plated in polyhema coated 96-well plates and cell viability was measured using a colorimetric assay at the indicated time points. D. Control and ESE3 knock-down LNCaP cells were plated in polyhema and assayed as described above. E. VCaP cells were transfected with siRNAs against ERG or EZH2 and plated in polyhema after 24 h. Cell viability was measured as described above. F. Parental and ERG-expressing (ERG-18) LNCaP cells were transfected with siRNAs against EZH2 and assayed as indicated above. G. Parental and ERG-expressing (ERG-18) LNCaP cells were transfected with full length Nkx3.1 expression vector and assayed as indicated above. H. Parental (sh-) and ESE3 knock-down (sh-7) LNCaP cells were transfected with full length Nkx3.1 expression vector and assayed as indicated above. Data are presented as mean ± SD of triplicate experiments. *, p<0.01; **, p<0.001.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Over Expression, Knockdown, Migration, Control, Expressing, Clone Assay, Colorimetric Assay, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: ESE3 controls expression of EZH2 and Nkx3.1 in normal prostate epithelial cells. ERG over-expression or loss of ESE3 leads to abnormal expression of these key genes and promotes cell transformation.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Expressing, Over Expression, Transformation Assay