Invadopodia elongation and chemoinvasion assay. (A) Schematic diagram of the chemoinvasion assay. Blue lines correspond to the x–y planes shown in B. (B) HCT116 cells in the chemoinvasion assay. Fluorescent Matrigel, actin stained with phalloidin-Cy3, cortactin, and a merged image of actin and Matrigel are shown. (top) x–y projections presenting the cell on the top of the filter. (middle) x–y projections presenting the cell below the focal plane of the filter. Arrows and arrowheads indicate a short and long protrusion, respectively. On the merged picture, the white rectangular corresponds to the longitudinal cut shown in the bottom panels. (bottom) x–z projections showing longitudinal cut through the cell at the level of the protrusions. (C) Effect of the metalloprotease inhibitor GM6001 on the formation of invadopodia. (left) Gelatin assay shows the percentage of cells that degraded the gelatin. (right) Chemoinvasion assay shows the percentage of cells that formed mature invadopodia. The results were normalized to the cells treated with DMSO. Light gray bars correspond to the cells treated with 10 µM GM6001. Dark gray bars correspond to control DMSO-treated cells. Error bars indicate SEM. *, P < 0.001; paired t test. Bars, 5 µm.