Fig. 3. RNASE4 induces prostate cancer cell proliferation and phosphorylation of AKT and S6.

a, b Exogenous RNASE4 (1 μg/ml) stimulates cell proliferation (a) and cell cycle progression (b) of DU145 cells cultured in the presence of 2% FBS. Cell numbers were determined by MTT assay. Cell cycle status was determined by flow cytometry after 24 h incubation with RNASE4. c Time course of AKT and S6 phosphorylation of serum-starved DU145 cells by RNASE4 (1 µg/ml). Left panels, immunoblots; right panels, quantification of p-AKT and p-S6 normalized to total AKT and S6 by Image J analysis. d Effect of AKT inhibitor MK-2206, PI3K inhibitors Wortmannin and LY294002, and mTOR inhibitor Rapamycin on RNASE4-induced phosphorylation of AKT and S6. Cells were pre-incubated with the inhibitors at the indicated concentrations for 1 h prior to be stimulated by 1 µg/ml of RNASE4 for 5 min. Top panels, immunoblots; bottom panels, Image J quantification of p-AKT and p-S6 normalized to total AKT and S6. e Effect of AKT inhibitor MK-2206, PI3K inhibitors Wortmannin and LY294002, and mTOR inhibitor Rapamycin on RNASE4-induced cell proliferation. DU145 cells were serum-starved overnight, incubated with the inhibitors for 1 h, and then treated with RNASE4 (1 µg/ml) for three days. Cell numbers were determined by MTT assay. Data shown are from a representative experiments in triplicates of 3 independent repeats. Error bars indicate SEM. **p ≤ 0.01, by unpaired two-tailed Student’s t test.