Figure 4.

WWP2 is required for tumorigenicity of cells. (a) DU145 cells were stably transfected with either retroviral-based control shRNA or two different WWP2 shRNAs. The expression levels of various proteins were analysed by immunoblotting with their respective antibodies. Actin was used as a loading control. (b) DU145 clones stably expressing control shRNA or WWP2 shRNAs were seeded and analysed for proliferation. The data shown are derived from four independent experiments (±s.d., P < 0.01, compared with cells expressing control shRNA; Student’s t-test). (c) DU145 cell lines stably expressing control shRNA or WWP2 shRNA were tested for anchorage-independent growth in a soft-agar colony assay. Viable colonies after 3 weeks were counted and the data (±s.d.) from three independent experiments were presented (P < 0.01, compared with cells expressing control shRNA; Student’s t-test). (d) Puromycin-resistant BPH1 prostate epithelial cells stably expressing either WWP2 wild type or the C838A mutant were established and the expression levels of PTEN and WWP2 were detected by the indicated antibodies. (e) A BPH1 parental cell line and BPH1-WWP2 wild-type or C838A mutant cells were analysed for proliferation in a similar way to that described in b. The data shown are derived from four independent experiments (±s.d., P < 0.05, compared with BPH1 parental cell line; Student’s t-test). (f) A non-transformed BPH1 cell line along with WWP2 wild-type- or C838A-mutant-expressing BPH1 cells were tested for anchorage-independent growth in a similar way to that described in c, and the data (±s.d.) were presented as summary of three independent experiments (P < 0.05, compared with BPH1 parental cell line; Student’s t-test). (g) DU145 stable clones expressing control shRNA or WWP2 shRNA alone or in combination with PTEN siRNA were analysed for proliferation in a similar way to that described in b. The data shown are derived from four independent experiments (±s.d., P < 0.01, compared with cells expressing control shRNA; Student’s t-test). (h) DU145 stable cell lines expressing control shRNA or WWP2 shRNA alone or in combination with PTEN siRNA were tested for anchorage-independent growth in a similar way to that described in c and the data (±s.d.) were presented as a summary of three independent experiments (P < 0.05, compared with cells expressing control shRNA; Student’s t-test). (i) Control shRNA- or WWP2 shRNA-expressing DU145 stable cells (5×10⁶) were subcutaneously injected into nude mice and the tumour volumes were measured three times per week (±s.d., n = 5, P < 0.05, compared with cells expressing control shRNA; Student’s t-test). Uncropped images of blots are shown in Supplementary Fig. S4.