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. 2012 Jul 16;1(7):e20. doi: 10.1038/oncsis.2012.20

Figure 3.

Figure 3

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.