Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: ETV4 expression levels in human prostate cell lines. ( a ) ETV4 expression level (normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH )) in comparison with normal human prostate (HmRNA) was assessed by quantitative real-time (qRT)–PCR in a nonmalignant human prostate cell line (RWPE) and in human cancer prostate cell lines (DU145, PC3, LNCaP and VCap). ( b ) ETV4 expression level assessed by western blot analysis in human prostate cell lines expressing ETV4 mRNA. ( c and e ) ETV4 expression level (normalized to the housekeeping gene GAPDH ) assessed by qRT-PCR before and after the doxycycline (Doxy) induction of DU145 ( c ) and PC3 ( e ) cell lines stably transduced with vectors expressing the indicated shRNA. ( d and f ) ETV4 expression level assessed by western blot analysis before and after the Doxy induction of DU145 ( d ) and PC3 ( f ) cell lines stably transduced with vectors expressing the indicated shRNA. CTL (irrelevant shRNA); sh3 (anti-ETV4 shRNA 3); sh65 (anti-ETV4 shRNA 65); see Materials and methods for details.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Expressing, Comparison, Quantitative RT-PCR, Western Blot, Stable Transfection, Transduction, shRNA

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: Effects of ETV4 silencing on the growth of DU145 and PC3 human PC cell lines. ( a and b ) Doxycycline-induced ETV4 silencing reduces the growth of DU145 ( a ) and PC3 ( b ) cell lines. Cell growth has been assessed by H 3 -thymidine uptake. At each time point, it is represented the ratio between initial and the actual c.p.m. (average of three experiments done in quadruplicate). Circles represent the cell lines stably transduced with a vector expressing an irrelevant shRNA; squares represent the cell lines stably transduced with a vector expressing the anti-ETV4 shRNA 65 (sh65); and triangles represent the cell lines stably transduced with a vector expressing the anti-ETV4 shRNA 3 (sh3). Full symbols connected by continuous lines represent the culture with doxycycline (with reduced ETV4 expression levels). Empty symbols connected by dotted lines represent the culture without doxycycline (normal ETV4 expression levels). ( c ) Doxycycline-induced ETV4 silencing reduces the substrate-independent growth of DU145 ( P <0.01) and PC3 cell lines ( P <0.05). Each bar represents the ratio between the number of colonies from plates with doxycycline and the number of colonies from plates without doxycycline. S.d.'s are shown. CTL (irrelevant shRNA); sh3 (anti-ETV4 shRNA 3); sh65 (anti-ETV4 shRNA 65); see Materials and methods for details. ( d ) Monolayer of stably transduced DU145 (on the left) and PC3 (on the rigth) cells after culture for 5 days with doxycicline was scored and then cultured in medium for the time showed in the picture and in presence of mitomycin C. Cell migration into the wound was examined by phase contrast microscopy (pictures of a representative experiment are shown).

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, shRNA, Cell Culture, Migration, Microscopy

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: ETV4 overexpression and its effects on human RWPE. ( a ) ETV4 expression level (normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH )) by quantitative real-time (qRT)–PCR in RWPE cells transfected with ETV4 expression vectors FL and TME (see Materials and methods for details) compared with untransfected (untr) and mock-transfected (LUC) RWPE cells. ( b ) ETV4 expression level assessed by western blot analysis in mock (LUC), FL and TME-transfected RWPE cells. ( c ) ETV4 overexpression increases the growth of RWPE cell line (assessed by H3-thymidine uptake). At each time point it is represented the ratio between initial and the actual c.p.m. (average of three experiments done in quadruplicate). Empty circle: untransfected RWPE; full circle, mock transfected (LUC) RWPE; diamond, FL-transfected RWPE; and cross, TME-transfected RWPE. ( d ) ETV4 overexpression increases the substrate-independent growth of RWPE cell lines (UNTR or LUC vs TME or FL RWPE, P <0.05). Each bar represents the average number of colonies (three experiments done in triplicate). S.d.'s are shown. ( e ) Upper panels: monolayer of mock transfected, FL-transfected RWPE cells was scored and then cultured in medium for 48 h in the presence of mitomycin C. Cell migration into the wound was examined by phase contrast microscopy (pictures of a representative experiment are shown). Lower panel: ETV4 overexpression increases the migration of RWPE cell lines (LUC vs TME-RWPE or FL-RWPE, P <0.03): each bar shows the difference between the width of wound at time 0 and that after 48 h normalized by the width of wound at time 0. Values are expressed as mean+s.d. (three experiments in duplicate). ( f ) ETV4 overexpression increases the migration through matrigel of RWPE cell lines (LUC vs TME-RWPE or FL-RWPE, P <0.05). Cells that migrated out of the chamber were stained and counted. Each bar shows the number of migrating cells normalized to that of the mock-transfected cells. The mean+s.d. of three experiments done in triplicates is shown.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Cell Culture, Migration, Microscopy, Staining

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: ETV4 silencing reduces the growth of DU145 and PC3 cell in xenograft model. ( a ) Tumors derived from DU145 cells stably transduced with doxycycline-inducible vectors expressing either an irrelevant shRNA (CTL) or the anti-ETV4 shRNA 65 and injected in athymic nude mice (representative experiment). Tumors have been excised from the immunodeficient mice treated with doxycycline after 8 weeks from the injection. ( b ) Each bar shows the ratio between the average weight of tumors recovered from doxycycline-treated mice and the average weight of tumors recovered from untreated mice. CTL (irrelevant shRNA); sh65 (anti-ETV4 shRNA 65); see Materials and methods for details.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Derivative Assay, Stable Transfection, Transduction, Expressing, shRNA, Injection

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: The effect of ETV4 silencing and of ETV4 overexpression on the cell cycle. ( a and b ) Cell cycle analysis carried out in PC3 ( a ) and RWPE ( b ) cells after treatment with 10 mℳ hydroxyurea for 14 h followed by culture in normal medium. The analysis of the cell cycle phases was performed at 0, 4, 8 and 24 h. The percentage of cells present in each specific phase of the cell cycle at each time point is shown. Filled diamond, cells in G1-phase; empty triangles, cells in S-phase; and dashed square, cells in G2-phase. PC3-CTL (PC3 cells stably transduced with an irrelevant shRNA); PC3-sh3 (PC3 cells stably transduced with anti-ETV4 shRNA 3); RWPE LUC, RWPE TME and RWPE FL (RWPE cells transfected with mock, TME and FL vectors, respectively). ( c ) Fold changes of expression levels (normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase) of genes known to be involved in cell cycle in PC3-sh3 (PC3 cells transduced with anti-ETV4 shRNA 3) compared with PC3-CTL (PC3 cells transduced with irrelevant shRNA) and RWPE cells transfected with FL-ETV4 plasmids compared with LUC-transfected RWPE cells. Fold changes are plotted on a Log 2 scale. ( d ) Expression (western blot analysis) of p21 (Waf1/Cip1) in PC3-sh3 compared with PC3-CTL and RWPE cells transfected with mock and FL-ETV4 plasmids.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Over Expression, Cell Cycle Assay, Stable Transfection, Transduction, shRNA, Transfection, Expressing, Western Blot

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: ETV4 overexpression induces EMT in the RWPE. ( a ) Representative microphotographs of RWPE cells are shown. Almost all mock-transfected RWPE cells displayed a rounded epithelial cell shape with rare spindle-shaped cells. A large fraction of cells RWPE transfected with FL and TME vectors displayed a spindle-like shape. ( b ) Fold changes of expression levels (normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase) of epithelial (E-cadherin) and mesenchymal (N-cadherin, vimentin and cadherin 11) markers in RWPE cells transfected with FL-ETV4 and TME-ETV4 plasmids compared with mock-transfected RWPE cells. Fold changes are plotted on a Log 2 scale. ( c ) Expression assessed by western blot analysis of epithelial (E-cadherin and zonula occludens) and mesenchymal (N-cadherin and vimentin) markers in RWPE cells transfected with mock (Luc), FL-ETV4 and TME-ETV4 plasmids. ( d ) Analysis by confocal microscopy of E-cadherin and of N-cadherin in RWPE cells transfected with mock, FL-ETV4 and TME-ETV4 plasmids. The luciferase-transfected RWPE cells do not express the mesenchymal marker protein N-cadherin and the epithelial marker protein E-cadherin is expressed and localized on the plasma membrane. In RWPE cells overexpressing ETV4, either TME or FL, the E-cadherin is reduced and has migrated from the plasma membrane to the cytoplasm; the N-cadherin is upregulated and it appeared in both the cytoplasm and plasma membrane.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Over Expression, Transfection, Expressing, Western Blot, Confocal Microscopy, Luciferase, Marker, Clinical Proteomics, Membrane

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: The effect of ETV4 overexpression on the expression of genes involved in EMT in the RWPE. ( a ) Expression level (normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase) of transcription factors associated with EMT in RWPE cells transfected with mock, FL and TME plasmids. ( b ) ETV4 expression level assessed by western blot analysis of some transcription factors (TWIST1, ZEB1, SNAIL1 and SLUG1) associated with EMT in RWPE cells transfected with mock, FL and TME plasmids.

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Over Expression, Expressing, Transfection, Western Blot

Journal: Oncogenesis

Article Title: Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition

doi: 10.1038/oncsis.2012.20

Figure Lengend Snippet: Sequences of oligonucleotides used for quantitative RT–PCR

Article Snippet: Proteins were detected by the following primary antibodies: ETV4 monoclonal antibody (Abnova, Atlanta, GA, USA); actin (Sigma-Aldrich, St Louis, MO, USA); vimentin, twist, E-cadherin, N-cadherin and Snail1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Zeb1 Zo-1 and Slug (Cell Signaling, Danvers, MA, USA).

Techniques: Sequencing