Figure 6.
Figure 6. The elongation of invadopodia relies on lamellipodial and filopodial machinery. (A and B) Immunofluorescence analysis of HCT116 cells in the chemoinvasion assay: spatial distribution of lamellipodial and filopodial markers in mature invadopodia. (left) x–y projections of the cell at the focal plane of the filter. The image is merged, showing actin revealed by phalloidin-Cy3 and the specified ABPs (green). Insets show the x–y projections of the ventral surface of the cell at the focal planes below the filter. Arrows indicate invasive protrusions. (right) z projections of the indicated protrusion. A, actin (red); ABP, ABP as specified in the left panels (green); M, merge. (A) ABPs associated with lamellipodia revealed by immunostaining (cortactin and p34 subunit of Arp2/3 complex) or by expression of GFP fusion proteins (VASP and α-actinin). (B) ABPs associated with filopodia visualized by immunostaining (myosinX) or by expression of GFP fusion proteins (fascin, T-fimbrin, and mDia2). For the visualization of fascin, the nonphosphorylatable mutant (S39A) was used. (C and D) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in MDA-MB-231 cells in the chemoinvasion assay. (C, left) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (right) mRNA expression levels of mDia2 in the cells transfected with two independent siRNA against mDia2 and control siRNA. β-Actin mRNA levels were used as a loading control. (D) Repartition of the protrusions according to their length in cells transfected with siRNA as indicated. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). The dashed line (50%) is shown to compare the distribution of protrusion lengths between control and siRNA-treated cells. *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (E) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in HCT116 cells in the native BM model. Repartition of the protrusions according to their length transfected with siRNAs is shown. Light gray bars show short protrusions (<2.5 µm). Black bars show long protrusions (>2.5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method. (D and E) Error bars indicate SEM. Fas, fascin; myoX, myosinX; T-Fimb, T-fimbrin. Bars: (A and B, left) 5 µm; (A and B, right) 1 µm.

The elongation of invadopodia relies on lamellipodial and filopodial machinery. (A and B) Immunofluorescence analysis of HCT116 cells in the chemoinvasion assay: spatial distribution of lamellipodial and filopodial markers in mature invadopodia. (left) x–y projections of the cell at the focal plane of the filter. The image is merged, showing actin revealed by phalloidin-Cy3 and the specified ABPs (green). Insets show the x–y projections of the ventral surface of the cell at the focal planes below the filter. Arrows indicate invasive protrusions. (right) z projections of the indicated protrusion. A, actin (red); ABP, ABP as specified in the left panels (green); M, merge. (A) ABPs associated with lamellipodia revealed by immunostaining (cortactin and p34 subunit of Arp2/3 complex) or by expression of GFP fusion proteins (VASP and α-actinin). (B) ABPs associated with filopodia visualized by immunostaining (myosinX) or by expression of GFP fusion proteins (fascin, T-fimbrin, and mDia2). For the visualization of fascin, the nonphosphorylatable mutant (S39A) was used. (C and D) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in MDA-MB-231 cells in the chemoinvasion assay. (C, left) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (right) mRNA expression levels of mDia2 in the cells transfected with two independent siRNA against mDia2 and control siRNA. β-Actin mRNA levels were used as a loading control. (D) Repartition of the protrusions according to their length in cells transfected with siRNA as indicated. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). The dashed line (50%) is shown to compare the distribution of protrusion lengths between control and siRNA-treated cells. *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (E) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in HCT116 cells in the native BM model. Repartition of the protrusions according to their length transfected with siRNAs is shown. Light gray bars show short protrusions (<2.5 µm). Black bars show long protrusions (>2.5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method. (D and E) Error bars indicate SEM. Fas, fascin; myoX, myosinX; T-Fimb, T-fimbrin. Bars: (A and B, left) 5 µm; (A and B, right) 1 µm.

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