SphK1 and Androgen Signaling Figure 4. DHT stimulates SphK1 activity in a PI3 kinase-dependent manner in LNCaP cells. A, overnight serum deprived LNCaP cells were incubated under 5% CSS conditions and treated with 10 nM DHT for the indicated times. SphK1 activity was quantified and expressed as percent of untreated cells. Columns, mean of at least six independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; **, P,0.01; ns, non significant. B, overnight serum deprived LNCaP cells were incubated with 10 nM DHT for 45 min in presence or not of 2.5 mM SKI- 2 or 200 nM wortmannin (WT). Cell lysates were assayed for phospho-Akt and Akt expression by Western blot analysis. Similar results were obtained in three independent experiments (top). Overnight serum deprived LNCaP cells were incubated under 5% CSS conditions and treated with or without 10 nM DHT and 200 nM wortmannin (WT) for 45 min, and essayed for SphK1 activity (bottom). Columns, mean of at least five independent experiments; bars, SD. The P values between the means are as follow: **, P,0.01; ns, non significant. C, overnight serum deprived LNCaP cells were incubated in FBS or in CSS conditions for 6 days with or without 200 nM wortmannin (WT) or 1 mM LY294002 in presence or not of 10 nM DHT as indicated. Cell proliferation was determined by MTT assay and expressed as percent of control at the beginning of the experiment (Day 0). Columns, mean of at least eight independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; ns, non significant. D, overnight serum deprived LNCaP cells were incubated in FBS or in CSS conditions with or without 2.5 mM of SKI-2 or F-12509a in presence or not of 10 nM DHT for 24 h (top), 48 h (middle) or 6 days (bottom). Secreted PSA level was quantified 24 h after the indicated treatment (top). DNA synthesis and MTT-based cell proliferation measurement were expressed as percent of control at the beginning of treatment, 2 and 6 days, respectively. Columns, mean of at least five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; **, P,0.01; or ns, non significant. doi:10.1371/journal.pone.0008048.g004 PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e8048