Induction of Tks5 is required for the promotion of invasive activity. (A and B) A549 pulmonary carcinoma cells cultured in the presence of 5 ng/ml TGF-β for the indicated times (A) or 72 h (B) were subjected to quantitative RT-PCR analysis of SH3PXD2A mRNA (A) or to immunoblot analysis with the indicated antibodies (B). Quantitative data are means ± SD from three independent experiments. (C) Representative images of A549 cells cultured in the absence or presence of TGF-β for 48 h. The cells were stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei. Arrowheads indicate invadopodia-like structures showing Tks5 accumulation. Pixel intensities on the traversing dashed line were measured using LAS AF software and shown in a boxed area (arbitrary units). Bars, 50 µm. See also Video 10 for live imaging. (D) A549 cells transfected with control (Ctr) or Tks5 siRNAs were replated in Matrigel chambers and assayed for invasive activity in the absence or presence of TGF-β. Cells that had invaded through the Matrigel at 18 h after plating were stained with crystal violet (top), and those in three different sampling areas were counted. Bar, 200 µm. Quantitative data are means ± SD from three independent experiments. See also Fig. S3 A for immunoblot. (E) Experimental protocol for Matrigel invasion assays with RAW264.7 macrophages. (F–H) Control RAW264.7 macrophages (F) or those harboring Dox-sensitive constructs for hTks5-WT (G) or hTks5-ΔPX (H) were subjected to invasion assays as in E. Cells that invaded through the Matrigel were stained with crystal violet (F), and the area occupied by the invaded cells in three different sampling regions in a single chamber was quantified. Bar, 200 µm. The minus sign indicates no stimulus added to the culture. Data are means ± SEM for six independent experiments. *, P < 0.02; **, P < 0.002 (Student’s t test).