Figure 1

PSAP gene silencing decreases metastatic PCa cell adhesion to basement membrane proteins. (A) PSAP over-expression in metastatic prostate cancer cell lines. Equal amount of cell lysates or supernatants from PC-3, DU-145, and normal prostate epithelial (Pr.Ep) cells were subjected to western blotting with anti-PSAP and saposin C antibodies. (B) Stable PSAP down-modulation was accomplished by short-hairpin RNA targeted at PSAP gene. PC-3 and DU-145 metastatic PCa cell lines were stably transfected with a G418-resistant vector containing a shRNA sequence specific for human PSAP or a scrambled control sequence. Total RNA was extracted for RT-PCR (top). GAPDH transcript was used as an internal control for RNA loading. Cell lysates and culture supernatants were subjected to immunoblotting with PSAP antibody. GAPDH antibody was used for protein loading. (C and D) PC-3 and DU-145 cell adhesion to ECM proteins was examined by seeding 1.5 × 10⁴ cells per well in 96-well plates pre-coated with 10 μg/ml fibronectin (FN) or laminin (LN). After 2 h incubation, adhered cells were fixed and stained with toluidine blue. Cells were photographed and counted from ten random fields at 100 × magnification. Columns, mean of three independent samples run together; bars, ± SEM, p < 0.0001, ANOVA was used to compare PSAP-KD and control clones. Each experiment was repeated three times independently. C1 and C3 in PC-3 and C9 and C13 in DU-145 were control clones (shRNA-scrambled vector) and P5 and P16 in PC-3 and P15 and P32 in DU-145 were PSAP-KD clones (shRNA-PSAP).