Figure 7

Knockdown of CXCL12 impaired SLUG-mediated MMP9 expression and prostate cancer cell migration. (A) qPCR analysis of MMP9 expression in PC3 cells overexpressing SLUG and CXCL12 shRNAs. PC3 stable cell lines overexpressing SLUG (or vector) and control shRNA (shCtr) or CXCL12 shRNA (CXCL12sh1 and CXCL12sh2) were proceeded to RNA extraction and cDNA synthesis. MMP9 transcript in these cell lines was analyzed by qPCR. (B) Cell migration assay in PC3 cells stably overexpressing SLUG. PC3 cells expressing pMig-Slug or pMig (vector) were seeded in 12-well plates (15 × 10⁴ cells per well). After cells formed a confluent monolayer, scratches were performed using a 100 μl tip. Twenty-four hours after scratching, the cells were examined for closure of scratch under the microscope and images were captured. Quantification of cell migration was done by measuring the distance between 4 random points within the wound edge in three replicate experiments. (C) Cell migration assay in PC3 cells stably overexpressing SLUG and CXCL12 shRNAs. PC3 stable cell lines overexpressing SLUG were infected with control shRNA or CXCL12 shRNA lentiviruses, as indicated. Closure of scratch was examined under the microscope 24 hr after scratching. Quantification of cell migration was done by measuring the distance between 4 random points within the wound edge. Slug-mediated cell migration was diminished in PC3 cells expressing CXCL12 shRNA.