Mouse GFP–Hic-5 expression in human MCF10A cells results in increased matrix degradation, motility, and invasion. (A) Western blot analysis shows expression levels of GFP, and mouse GFP–Hic-5–overexpressing cells have increased α smooth muscle actin. GFP–Hic-5 can be efficiently depleted with two independent siRNAs specific for mHic-5. Molecular mass standards are indicated next to the gel blots in kilodaltons. (B) GFP and GFP–Hic-5 cells plated on fluorescent 488 gelatin. GFP–Hic-5–expressing cells have the ability to degrade matrix, which is reversed with mHic-5 siRNA. Bar, 20 µm. (C) Quantitation of the percentage of cells associated with matrix degradation. (D) Quantitation of the area of matrix degraded per cell area. (E) Modified Boyden chamber assay shows that GFP–Hic-5 cells have increased motility that is reversed with mHic-5 siRNA. (F) GFP–Hic-5–expressing cells demonstrate increased invasion through Matrigel compared with GFP-expressing cells that is reversed upon knockdown of GFP–Hic-5. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Breaks in the x axis indicate separate sets of experiments, each n = 3. (G) GFP–Hic-5 localizes to the ring structure of invadopodia surrounding an actin core. Actin and cortactin colocalize to the core structure of invadopodia. Representative line profile plots demonstrate the distribution of GFP–Hic-5 to the ring, and actin and cortactin to the core of invadopodia. Error bars represent the standard error of the mean. Bars: (main panels) 10 µm; (insets) 1 µm.