Figure 5

Down-regulation of cathepsin D expression and activity decreased migration and invasion in PSAP-KD cells. (A) PSAP down-modulation reduced CathD expression and activity. Total RNA was subjected to RT-PCR using specific primers for CathD. Cell lysates and culture supernatants were prepared from parallel dishes and analyzed by immunoblotting with an anti-CathD monoclonal antibody which recognizes proCathD (P), intermediate CathD (I), and mature CathD (M). (B) Whole cell extracts were also assayed for CathD enzymatic activity using a kit with a fluorimetric substrate. The enzymatic activity of CathD was calculated as units/mg total protein. Columns, mean of three independent samples run together; bars, ± SEM. ANOVA was used to examine the significance of the data (p < 0.0001) comparing PSAP-KD clones versus control clones. (C) Transient down-modulation of CathD and its effect on PSAP expression. Control clones of PC-3 and DU-145 cell lines were transiently transfected with specific CathD- or control-siRNA oligos. After 48 h, cell lysates were analyzed for CathD, PSAP, and saposin C expression by immunoblotting. (D) Migration and invasion assays were performed on parallel-transfected tissue culture plates as described in the legend to Fig. 2. Columns, mean of three independent samples run together; bars, ± SEM, p < 0.0001, Two-sample t-tests with Satterthwaite corrections were used to compare CathD-siRNA versus scrambled-siRNA oligos transfected cells. Similar results were obtained from three independent experiments.