Figure 4. Sortilin depletion in PC3 cells enhances progranulin levels and downstream biological responses.

A, PC3 cells stably depleted of endogenous sortilin were generated by transfecting the pRS-shRNA-control (scrambled shRNA) or pRS/shSORT plasmids using the TransIT-Prostate Transfection kit. Cells were selected in medium supplemented with 2 μg/mL of puromycin. After selection, pools of sortilin-depleted PC3 cells were tested for sortilin expression by immunoblot using anti-sortilin monoclonal antibodies (R&D Systems). Densitometric analysis is expressed as arbitrary units. B, Progranulin expression levels in cell lysates and conditioned media as detected by immunoblot using anti-progranulin polyclonal antibodies as previously described (6, 7). Blots (A and B) are representative of 2 independent experiments. Migration (C), wound healing (D), proliferation (E), and anchorage-independent growth (F) in soft-agar were performed as described in Materials and Methods. In soft-agar assays, only colonies of more than 150 μm were counted. Data are the average of 3 independent experiments run in triplicates ± SD. **, P < .01; ***, P < .001.