SphK1 and Androgen Signaling Figure 9. The reversability of the neuroendocrine-like phenotype by exposure to normal medium is associated with down- regulation of SphK1 activity. A, LNCaP cells were left under CSS conditions for the indicated times (from 0 to 42 days). Cells were then collected and plated in CSS for 2 days to ensure complete adhesion before treatment with 5% FBS, 5% CSS with or without 10 nM DHT for 6 days (left panel) or 24 h (right panel). Cell proliferation was expressed as percent of control at the beginning of the 6 days treatment (A, left panel). Secreted PSA level was quantified 24 h after treatment with 5% FBS, 5% CSS with or without 10 nM DHT of LNCaP cells (A, right panel). Columns, mean of five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; or ns, not significant. B, LNCaP cells were left in CSS conditions for 42 days and then cultured back for an additional 21 days in 5% FBS. Cells were then collected and plated in 5% FBS conditions for 2 days to ensure complete adhesion before treatment with 5% FBS, 5% CSS with or without 10 nM DHT for 6 days (left panel) or 24 h (right panel). Cell proliferation was expressed as percent of control at the beginning of the 6 days treatment (B, left panel). Secreted PSA level was quantified 24 h after treatment with 5% FBS, 5% CSS with or without 10 nM DHT of LNCaP cells (B, right panel). Columns, mean of five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; **, P,0.01; or ns, not significant. C, LNCaP cells were cultured under CSS conditions for 42 days and then cultures for 21 days in 5% FBS or 5% CSS as indicated, then SphK1 activity was assessed. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P,0.001. doi:10.1371/journal.pone.0008048.g009 Isoton II (Coulter Corp., Miami, FL), and 200 ml duplicates were counted in a model Z1 Coulter particle counter and averaged. Measurement of DNA Synthesis At the indicated times, cultures were pulsed with 1 mCi of [3H]- thymidine for 6 h and radioactivity incorporated into trichlor- oacetic acid-insoluble material measured as previously described [55]. Western Blot Analysis and Antibodies Western blotting was performed as previously described [54]. Rabbit anti-SphK1 (gift from Dr Pitson, IMVS, Adelaide, Australia), rabbit anti-Akt (Cell Signaling Technology # 9272), rabbit anti-Akt/phospho Ser473 (Cell Signaling Technology # 9271), mouse anti-CgA (Ventana Medical Systems), and mouse anti-FLAG (Sigma, clone M2) were used as primary antibodies. Proteins were visualized by ECL detection system (Perbio Science) using anti-rabbit or anti-mouse HRP-conjugated IgG (Bio-Rad, Marnes La Coquette, France). Equal loading was confirmed by probing the blots with the mouse anti-b-actin (Sigma, clone AC40) or anti-tubulin (Sigma, clone DM1A). Sphingosine Kinase-1 Assay SphK1 activity was quantified as described previously [52], and determined in the presence of 50 mM sphingosine, 0.25% Triton X-100 and [c-32P]-ATP (10 mCi, 1 mM) containing 10 mM MgCl2. The labelled S1P was separated by thin layer chromatog- raphy on silica gel 60 with 1-butanol/ethanol/acetic acid/water (80:20:10:10, v/v) and visualized by autoradiography. Activity was expressed as picomoles of S1P formed/min/mg of protein. Quantitation of Secreted PSA and NSE Levels Cells were seeded at an initial density of 7,800 and 4,200 cells/ cm2 for LNCaP and PC-3 respectively. After treatment, culture media were collected and centrifuged. Total PSA was quantified by the Abbott MEIA PSA assay (Abbott AxSYM systemH PSA total). NSE levels were determined by the TRACE (Time Resolved Amplified Cryptate Emission) technology, based on a non-radiative transfer of energy (Brahms NSE KryptorH). cAMP Assay The intracellular concentration of cAMP was carried out with the cAMP complete enzyme immunoassay kit from Assay designs PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e8048