SPRY2 deficiency–induced ROS increases nuclear accumulation of PTEN.
(A) DU145 and MEFs were analyzed for intracellular ROS using DCFDA dye in the presence and absence of 10% serum (*P < 0.01; n = 3, analyzed by Mann-Whitney test). (B) Intracellular ROS was detected using DCFDA dye and analyzed in 24-hour serum-starved DU145 cells following treatment with growth factors (FGF2 [10 ng/ml], IGF-1 [100 ng/ml]) along with respective inhibitors (FGFR inhibitor- BIBF1120 [0.4 μM], IGF-1R inhibitor, PPP [2 nM]) for 30 minutes. SS, serum starved. (*P < 0.01; n = 3, analyzed by Mann-Whitney test). (C) Representative immunofluorescence images of MEFs stained for Pten (green) and p21 (red) after 5 μM NAC treatment for 48 hours. Scale bars: 100 μm. (D) Nuclear extracts from DU145 cells treated with 5 μM NAC for 48 hours were analyzed by Western blot, and G1 cells were quantified (*P < 0.001; n = 3, analyzed by Mann-Whitney test). (E) Representative images and prostate weights of nude mice orthotopically injected with Nsi or SPRY2 KD (CL61) DU145 cells and treated with NAC. Scale bars: 0.5 cm. (*P < 0.01, number of mice = 7, analyzed by Dunnett’s multiple comparison test). Box and whisker plots show median (lines within boxes), interquartile range (bounds of boxes), and upper and lower range (whiskers). (F) Representative images of IHC for Ki67, PTEN, TP53, and p21 in indicated DU145 orthotopic tumors. Scale bars: 100 μm. All the Western blots were quantified using ImageJ, and the values represent relative immunoreactivity of each protein normalized to respective loading control. Data are presented as mean ± SEM (A, B, and D).