Ectopic expression of Hck provides fibroblasts the capacity to migrate through Matrigel. (A) Tet-Off MEF-3T3 cells expressing Hck isoforms together or separately that migrated through Matrigel transwells were counted as in the legend of Figure 2C. Results are expressed as mean ± SD of 3 experiments; parental cells were Hck-negative Tet-Off MEF-3T3 cells. (B) Photomicrograph showing nuclei (DAPI [4,6 diamidino-2-phenylindole]: blue), p59/61Hck^ca-GFP (green), and F-actin (phalloidin–tetramethyl rhodamine isothiocyanate: red); arrows indicate podosome rosettes located at the pore exit (dotted circles) of the transwell. p59/61Hck^ca-MEF-3T3 Tet-Off cells had more podosome rosettes after transmigration (24 hours, cells examined at the bottom face of the 8-μm pore transwells) than before (0 hours, cells examined at the top of transwells). (C) The cell migration capacity through Matrigel is correlated to the rate of podosome rosettes. The percentage of cells with podosome rosettes was quantified in 2 p59/61Hck^ca-MEF-3T3 Tet-Off cell clones (clones A and B; left panel) and invasion assays were performed in triplicate in 4 separate experiments (right panel); ***P < .001.