Figure 2. Mena and Mena^INV Promote Carcinoma Cell Invasion into a 3D Matrix and Enhance Stabilization and Degradation at Invadopodia.

(A) 3D Collagen invasion assay using MTLn3 cells expressing Mena or Mena^INV in the absence (white bars) and presence (gray bars) of macrophages. Error bars indicate SEM. *p < 0.005 compared to parental MTLn3 cells; ***p < 0.005 compared to MTLn3 Mena cells.

(B) 3D Collagen invasion assay using MTLn3 cells expressing Mena^INV in the presence of DMSO or 6 μM AG1478. Error bars indicate SEM.

(C) 3D reconstruction of MTLn3 EGFP-Mena cells transiently transfected with RFP-cortactin invading collagen I. Arrows indicate 3D protrusions (collagen I gel on bottom). Scale bar, 10 μm. Higher magnifications of the protrusion are shown on the bottom right; scale bar, 5 μm.

(D) Schematic diagram of the invadopodium assay. Images in (E) and (G) represent the plane of the matrix in this diagram.

(E) Immunofluorescence of MTLn3 cells using anti-Mena or anti-cortactin antibodies. Arrowheads indicate invadopodia. Scale bar, 10 μm.

(F) Matrix degradation by MTLn3 cells expressing EGFP, EGFP-Mena, or EGFP-Mena^INV. Bars represent the mean of two independent experiments and over 40 cells analyzed. Error bars indicate SEM. *p < 0.00005.

(G) Images of Texas Red-fibronectin matrix indicating degradation by invadopodia visible as black holes (arrowheads). Scale bar, 10 μm.

(H) Analysis of invadopodium lifetime of MTLn3 GFP-Actin cells transiently transfected with mCherry-Mena or mCherry-Mena^INV. Non-transfected cells on the same dish served as control. Error bars indicate SEM. *p < 0.005.

(I) Histogram showing the distribution of invadopodium lifetimes.