Analysis of the effects of osteopontin (OPN) expression on MMP-9 activity. A. OPN expression levels were measured in different PC3 cell lines (indicated below each lane) by immunoblotting analysis. Untransfected PC3 cells (lane 1), PC3 cells transfected with pCEP4 vector (lane 2), pSilencer 4.1-CMV neo vector (lane 3), and Scrambled RNA construct (lane 10) were used as controls. Basal level expression of OPN was observed in these cells. An increase in OPN expression was observed in PC3 cells transfected with full length (PC3/OPN; lane 4) and mutated (PC3/OPN (RGA); lane 5) OPN cDNA. Individual clones (denoted as C1) that express maximum level of OPN is used for the studies shown here (lanes 4 and 5). PC3 cells transfected with four different SiRNA constructs exhibited expression of different levels of OPN (lanes 6–9). Immunoblotting with an antibody to GAPDH was used as a loading control (lower panel in A). B and C: Analysis of MMPs activity in the conditioned media of different PC3 cell lines by gelatin zymography: Several individual clones (10–15) were isolated from PC3 cells transfected with different OPN constructs (full length, mutated (RGDΔRGA), and SiRNA). Conditioned media collected from clones that express maximum (Figure B, lanes 3–10; C1–C4 in PC3/OPN and PC3/OPN(RGA)) and reduced levels (Figure C, lanes 3–7, C1–C5) of OPN protein were used for zymogram analysis Untransfected PC3 cells were used as control (lane1 in B and C). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 1 in B and C). Gelatinolytic activities of both MMP-2 and MMP-9 are indicated by arrows (B and C). The results represent one of three separate experiments performed.