Figure 5

Analysis of osteopontin (OPN) expression and cell migration in Met-As and Db-S stable cell lines. (a) Detergent lysates were prepared from the indicated cell lines. Proteins were size-fractionated by 7.5% SDS-PAGE and transferred to a polyvinylidenedifluoride membrane and analyzed for OPN expression with antimouse antibody (AF808; R&D Systems, Minneapolis, MN, USA). OPN migrated as a doublet, presumably due to isoforms generated by different levels of glycosylation (arrows). Molecular mass standards are indicated on the left-hand side. The lowermost band present in all four lanes is due to nonspecific binding. (b) Relative OPN expression and (c) the fold change in OPN expression in Met-As cells and Db-S cells compared with their relevant vector-transfected control cell lines were determined in the blot in (a) using the TotalLab version 2.00 software package (NonLinear Dynamics, Ltd, Durham, NC, USA). (d) Migration assays were performed on the Db-S cells, Met-As cells, Db control cells, and Met control cells. Met-As cells showed a threefold decrease (P < 0.001) in the number of cells that migrated in comparison with the Met control cells. Db-S cells showed a 10-fold increase (P = 0.001) in migration when compared with the Db control cells.