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. 2013 Dec 23;203(6):1063–1079. doi: 10.1083/jcb.201306162

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© 2013 Monteiro et al.

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Figure 6. — WASH and exocyst complex are required for matrix remodeling and the invasive potential of breast tumor cells. (A) MDA-MB-231 cells treated with indicated siRNAs were plated on cross-linked FITC-labeled gelatin and the percentage of degradative cells (gray bars) and the degradation index (white bars) were calculated. Values are means ± SEM from at least three independent experiments scoring a total of 300–400 cells for each siRNA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with siNT-treated cells). (B) MDA-MB-231 cells treated with indicated siRNAs were quantified on the lower side of the Transwell filter after invasion through Matrigel. Values are mean ± SEM of normalized percentage from two to three independent experiments. *, P < 0.05; **, P < 0.01 (compared with siNT-treated cells). (C) 3D type I collagen circular invasion assay of MDA-MB-231 cells expressing Histone-2B-GFP treated with indicated siRNAs after 48 h. Dashed circles represent the collagen–cell interface at time 0. (D) Values represent mean invasion index ± SEM for the different cell populations from three independent experiments, normalized to cells treated with non-targeting siRNA (***, P < 0.001). (E) MDA-MB-231 cells treated with the indicated siRNAs were embedded in collagen I. Pericellular collagenolysis was detected using anti-Col1-^3/4C antibodies (in black in the inverted image). Nuclei were stained with DAPI (red). Bars, 50 µm. (F) Quantification of collagenolysis by MDA-MB-231 cells treated with the indicated siRNAs. Values are mean normalized degradation index ± SEM from three (siNT, siExo84d, and siMT1-MMP) or two independent experiments (siWASHd). n represents the number of cells analyzed for each cell population. ***, P < 0.001 (as compared with siNT-treated cells).