Figure 2: WIP and WIRE are necessary for invadopodium-mediated degradation.

(a) Stably infected MDA-MB-231 cells (shControl, shWIP, shWIRE and double shWIP/WIRE knock-down) were cultured on mouse peritoneal basement membrane (BM) (4 d), fixed, and stained for mouse type IV collagen (red), nuclei (DAPI, blue) and F-actin (green), and visualized by confocal microscopy. Serial optical sections were captured at 2-μm intervals. Bars: 20 μm. (b) Remaining type IV collagen in BM was measured in Z-projections and normalized to native membrane levels. (c) Cell distribution across the BM was calculated as the percentage of cells located at three different sectors of the stack: on top (grey), embedded in (black) and beneath the BM (white). (d) MDA-MB-231 or MCF-7 cells were cultured on mouse peritoneal BM (24 h), fixed and stained as in (a). Bars: 10 μm. Arrowheads indicate invadopodia. (e) Stably infected shControl, shWIP or shWIRE MDA-MB-231 cells were plated on rhodamine-gelatin-coated glass coverslips (red, 5 h), fixed and stained for F-actin (green) and cortactin (cyan). Bars: 10 μm (insets, 5 μm). (f) Quantification of invadopodium-forming cells (number of cells that form invadopodia), degrading cells (number of cells that degrade gelatin) and degraded area (calculated as degraded area/cell area) were normalized to control values. Data show mean ± SD of at least three independent experiments (N ≥ 75 cells/condition). *p < 0.05, **p < 0.01, ***p < 0.001 by 1-way ANOVA and Tukey’s post-hoc test (b and degraded area measurements in (f) or Chi-square test (c and f, for invadopodium-forming and degrading cells).