Figure 2.

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 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). 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).