Figure 5
Targeted siRNA-mediated inhibition of WT1 expression reduces PCa cell proliferation and blocks cell cycle progression. (a) Relative cell viability of 22Rv and LNCaP PCa cells measured via cell titer assay at 3 d post-transfection with WT1 siRNA or si-NC (20 nM). Validation of WT1 knockdown see Fig. S2B. Error bars = SD; n = 3; **p < 0.005. (b) Proliferation of 22Rv1 and LNCaP PCa cells (cell index) measured using the xCELLigence system post WT1 siRNA or si-NC transfection (20 nM). Validation of WT1 knockdown see Fig. S2C. Error bars = SD; n = 3. (c) Colony formation assay of 22Rv1 and LNCaP PCa cells 14–21 days post WT1 siRNA or si-NC (20 nM) transfection. Validation of WT1 knockdown see Fig. S2C. Error bars = SD; n = 3; **p < 0.005. (d) Flow cytometry cell cycle analysis of 22Rv1 and LNCaP PCa cells transfected with WT1 siRNA or si-NC (20 nM) for 72 h. Validation of WT1 knockdown see Fig. S1D. n = 3; *p < 0.05, **p < 0.005 relative to si-NC. (e) Western blot analysis of p21 (22Rv1 and LNCaP) and p53 (22Rv1) protein expression 72 h post-transfection of PCa cells with 20 nM WT1 siRNA or si-NC. β-actin is the loading control. Validation of WT1 knockdown see Fig. S2E. For full-length, non-cropped blots see Fig. S1B and S1C. n = 3. (f) Relative cell viability of 22Rv1 and LNCaP PCa cells measured via cell titer assay at 5 days post-transfection with WT1 siRNA or si-NC (20 nM), and 3 d post-17-AAG treatment (1 µM). Validation of WT1 knockdown see Fig. S2B. Error bars = SD; n = 3; **p < 0.005.
