PP2A activation suppresses tumorigenesis by increasing nuclear localization of PTEN.
(A) DU145 cells infected with control and PP2A-A–expressing viruses were treated with GSK3B siRNA for 24 hours and analyzed by Western blot and WST-1 assay for measuring cell proliferation (*P < 0.01; n = 3). (B) Representative immunofluorescence images of PP2A-A–expressing DU145 cells treated with GSK3B siRNA for 24 hours and stained for PTEN. Scale bars: 50 μm. (C) Western blot analysis of DU145 SPRY2 KD clones with stable expression of constitutively active GSK3B (S9A) treated with 5 nmol PP2A-A siRNA for 42 hours. (D) DU145 and MEF cells treated with 0.2 μM okadaic acid or 10 μM FTY720 for 12 hours were quantified for percentage of G1 cells (*P < 0.01; n = 3). (E) Representative images and PP2A activity measurement in s.c. xenografts of DU145 cells in nude mice treated with PP2A activator FTY720 (10 mg/kg/d i.p. injection) for 5 days. (*P < 0.01; n = 5). (F) Representative images and tumor burden of s.c. injected DU145 cells in nude mice treated as indicated. Red line indicates schedule of FTY720 treatment (10 mg/kg/d i.p. injection). (G) Representative IHC images and quantification of Ki67 (white), PTEN (gray), and p21 (blue) in DU145 s.c. xenografts treated as above. Scale bars: 50 μm. Box and whisker plots (E and G) show median (lines within boxes), interquartile range (bounds of boxes), and upper and lower range (whiskers). The values of Western blot represent relative immunoreactivity of each protein normalized to respective loading control. Data are presented as mean ± SEM and analyzed by Mann-Whitney test (A and D–F).