CXCL12 is required for Slug-mediated prostate cancer cell invasion. (A) Invasion assay of PC3 cells expressing pMig-Slug, or pMig (vector). Briefly, cells were seeded at 6 × 104 cells per well into 96-well plate from the Oris™ Cell Invasion Assay Kit, and incubated for ~16 hr at 37°C. Collagen I overlay was added to create a 3-D ECM environment for invasion. Cells were allowed to invade for 48 hr, and then stained with DAPI before images were captured (left panel). Total number of invaded cells were manually accounted (right panel). (B, C) Invasion assay of PC3 cells overexpressing SLUG and CXCL12 shRNAs. PC3 cell line overexpressing SLUG (pMig-Slug) and control shRNA (Ctr) or CXCL12 shRNA (Sh1, Sh2) were generated as shown in Figure 6C, and their invasion ability was examined as in (A), based on relative numbers of invaded cells (C).