ARF6 is required for matrix degradation and invasive migration of breast tumor cells through 3D type I collagen. (A) FITC-gelatin degradation. n, number of cells scored for each cell population treated with indicated siRNAs. ***, P < 0.001 (compared with siNT-treated cells). (B) Phalloidin-labeled MDA-MB-231 cell spheroids after 2 d in 3D collagen I (T2). Insets show spheroids immediately after embedding in collagen (T0). Bars, 200 µm. (C) Mean invasion area of spheroids at T2 normalized to mean spheroid area at T0 and to invasion of siNT spheroids set to 100 ± SEM. n, spheroid number. ***, P < 0.001. (D) MDA-MB-231 cells treated with indicated siRNAs embedded in collagen I and stained for cleaved collagen with Col1-^3/4C antibody (black in the inverted image). DAPI-stained nuclei are shown in red. Bar, 20 µm. (E) Collagenolysis by MDA-MB-231 cells (Col1-^3/4C antibody signal as in D). Values are mean normalized degradation index ± SEM. n, number of cells analyzed for each cell population. ***, P < 0.001 (as compared with siNT-treated cells).