Localization and role of intermediate filaments during invadopodia formation and elongation. (A) Gelatin degradation assay using MDA-MB-231 cells. (top) Localization of cytokeratins. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), cortactin, and cytokeratin filaments are shown. (bottom) Localization of vimentin. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), the Arp2/3 complex, and vimentin filaments are shown. Insets show higher magnification images of the boxed regions. (B) Chemoinvasion assay and localization of intermediate filaments in mature invadopodia in HCT116 cells. (top) Cytokeratin filaments. (bottom) Vimentin filaments. (left) x–y projections of the cell above the focal plane of the filter. Merged images of actin and intermediate filaments (green) are shown. Insets show x–y projections of the cell below the focal plane of the filter. Arrows and asterisks indicate protrusions shorter and longer than 5 µm, respectively. (middle) z projections of the indicated protrusions. A, actin (red); B, intermediate filaments (green); M, merged image. (right) Presence or absence of intermediate filaments in invasive protrusions according to their length. (C, top) Immunofluorescence analysis after depletion of vimentin filaments by siRNA in MDA-MB-231 cells. DAPI (left), vimentin (middle), and a merged image (right) are shown. si-control indicates cells treated with a scrambled siRNA; si-vim1 and si-vim2 indicate cells treated with siRNAs against vimentin. (bottom) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (D) Repartition of the protrusions according to their length in MDA-MB-231 cells treated with the indicated siRNA. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (E) Immunofluorescence analysis of MDA-MB-231 cells transfected with pEGFP (gfp) or pEGFP-vim1A (vim1A) plasmids. Actin revealed by phalloidin-Cy3 (left), GFP (middle), and vimentin filaments revealed by immunostaining for vimentin (right) are shown. (F) Repartition of the protrusions according to their length in MDA-MB-231 cells transfected with pEGFP or pEGFP-vim1A. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method. (B, D, and F) Error bars indicate SEM. Bars: (A, B [left], and E) 5 µm; (B, middle) 1 µm; (C) 10 µm.