Figure 2.

Effect of macrophage inhibitory cytokine-1 (MIC-1) overexpression on cell motility and invasion. Cells were seeded on noncoated or Matrigel-coated membranes for motility (a) and invasion (b) assays, respectively, and incubated for 22 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemoattractant. The cells that migrate through the membrane were stained and photographed under bright-field microscopy (magnification, × 10). The number of cells that migrated throughout the membrane was determined by averaging 10 random fields of view. The data are expressed as the number of cells per field of view and is the average of three independent experiments. Error bars indicate s.e. of the average (**P < 0.0005; *P< 0.05). (c) To show the paracrine effect of MIC-1 on the motility of other neighbor cells, we carried out a wound-healing assay, using conditioned media collected from PC-3-MIC-1, PC-3-vector, PC-3M-Con and PC-3M-siMIC-1 cells. In this experiment, cells in culture were monitored for their ability to migrate into a site of mechanical injury. Introduced wounds were photographed before and 10 h after incubation of cells in conditioned media. The results show that normal PC-3 cells incubated with conditioned media collected from PC-3-MIC-1 and PC-M-Con cells heal the wound quicker (**P < 0.0005) than cells incubated with media from PC-3-vectro or PC-3-siMIC-1 cells. The wound area covered after 10 h of incubation was determined as described in Supplementary Materials and Methods.