FIG 1.

Plk1 negatively regulates the Wnt/β-catenin pathway in human prostate cancer cells. (A) Genetic backgrounds of prostate cell lines used in this study. (B) Overexpression of Plk1 inhibits β-catenin signaling in RWPE1 cells. RWPE1 cells were infected with adenovirus expressing GFP or GFP-Plk1 for 48 h and harvested for immunoblotting (IB) with the antibodies indicated. (C) Knockdown of Plk1 activates Wnt/β-catenin signaling in MEFs from Plk1-inducible knockdown (iKD) mice. MEFs were harvested for IB after 96 h of treatment with doxycycline (Dox) (50 μg · ml⁻¹). (D) Vector-based siRNA targeting Plk1. PC3 or LNCaP cells were cotransfected with pBS/U6-Plk1 or the control vector and pBabe-puro at a ratio of 10:1. At 24 h posttransfection, the medium was changed, and 2 μg/ml puromycin was added to select for transfection-positive cells. After a 2-day selection with puromycin, floating cells were washed away, and the remaining attached cells were harvested for IB. (E) Depletion of Plk1 stabilizes β-catenin in various human prostate cancer cells. PC3, LNCaP, DU145, C4-2, 22RV-1, or MR49F cells were infected with lentivirus for 36 h to deplete Plk1 and harvested for IB. (F) Inhibition of Plk1 leads to activation of Wnt/β-catenin signaling. PCa cells were treated with the indicated concentrations of BI2536 for 16 h. (G) Cells were cotransfected with the TCF/LEF luciferase reporter vector or a noninducible TCF/LEF mutant luciferase vector and a constitutively expressing Renilla luciferase vector (internal control), followed by treatment with Wnt3a or BI2536. Luciferase activity is reported as relative activity (luciferase/Renilla) and represented as means ± standard deviations of data from 3 replicates. Wnt3a (as a positive control) is an established agonist of the Wnt/β-catenin pathway. *, P < 0.05; **, P < 0.01. (H) Plk1 inhibition enhances the levels of cytosolic and nuclear β-catenin. PC3 and LNCaP cells were treated with BI2536 or Wnt3a, followed by subcellular fractionation. (I to K) Representative confocal microscopy images of enhanced levels of cytosolic and nuclear β-catenin in PC3 and LNCaP cells treated with BI2536 or GSK46136A for 16 h (I) and quantified (J and K) (means ± standard errors of the means; n = 3 independent experiments, where 500 cells were counted in each group). **, P < 0.01. All Western blots are representative of data from three or more experiments.