Analysis of CD44 surface expression and migration in different PC3 cell lines. A-C. Analyses shown in Figures A-C were performed in the following PC3 cell lines: PC3 (lane 1), PC3/OPN (lane 2), PC3/OPN (RGA) (lane 3), and PC3/SiRNA (lane 4). Cells were surface- labeled with NHS-Biotin and lysates were immunoprecipitated with an antibody to vCD44 (V3-10) (A) or MMP-9 (C). Also, as an internal control, a monoclonal antibody to actin was added to vCD44 immunoprecipitation. Actin immunoprecipitation was used as an internal control for normalization. Expression of variant forms of CD44 was observed in PC3 cell lines. Immunoprecipitation with a species-specific non-immune serum did not show any protein bands in the immunoblotting analysis (data not shown). Shorter exposure blot for PC3 (lane 5) and PC3/OPN (lane 6) is shown. The immunoblot shown in A was stripped and blotted with an actin antibody (B). Detection of surface expression of MMP-9 by immunoblotting with streptavidin-HRP is shown in C. No changes in the surface levels of MMP-9 indicate that biotinylation reaction was equally efficient in the indicated PC3 cell lines. The results represent one of three experiments performed. D and E: Wound healing assay. Phase-contrast micrographs of PC3, PC3/OPN, PC3/OPN (RGA) and PC3/SiRNA cells at 0 h and 48 h are shown. Results represent one of three experiments performed. Statistical analysis is provided as a graph at 0 h and 48 h in panel E. A significant increase in the migration of PC3/OPN, PC3, and PC3/OPN (RGA) cells was observed as compared with PC3/SiRNA cells. *** p < 0.001 and ** p < 0.01 vs. PC3/SiRNA cells. For each cell line, two plates were used per experiment. Multiple uniform streaks (~7–9 streaks) were made on the monolayer culture for each cell line. The data are mean ± SEM of three experiments.