FIG. 6.

Impaired invasion in vitro of KO-NY72 cells. (A) Colocalization of mDia1 and pSrc in podosomes. WT-NY72 cells were shifted to the permissive temperature for 24 h, fixed, and stained either for pSrc, F-actin, and mDia1 or for cortactin and F-actin. Bar, 50 μm on the left and 20 μm on the right. (B) In situ zymography of WT-NY72 and KO-NY72 cells. WT-NY72 and KO-NY72 cells maintained at 40°C were plated on Oregon green 488-conjugated gelatin cross-linked glass coverslips and cultured at 37°C for 3 h. Cells were fixed and stained for actin and cortactin, and cells degrading fluorescent gelatin were counted. (C) Invasion in Matrigel. WT-NY72 and KO-NY72 cells were applied to the upper well of the Matrigel invasion chamber with or without serum in the lower well. After 24 h, Matrigel was removed and cells that passed through Matrigel and penetrated the membrane were stained and their number determined. The numbers of invading cells are shown as means ± SEM from three independent assays. Typical photomicrographs are shown on the right.