Figure 5.

Calpain 2 regulates invadopodia dynamics downstream of transforming v-Src kinase in MTLn3 breast cancer cells. (A) Control and calpain 2 knockdown MTLn3 cells expressing v-Src kinase were cultured on FN-coated glass coverslips and stained with anti-cortactin antibody and rhodamine phalloidin. Quantification of cortactin and actin containing invadopodia is expressed as the mean number of invadopodia per cell. Data shown are means ± SEM of three independent experiments. (B) GFP-cortactin was transfected into v-Src transformed MTLn3 control or calpain 2 siRNA lines. Cells were plated on FN-coated glass-bottomed dishes and analyzed by time-lapse fluorescence microscopy. Fluorescent images of v-Src transformed control or calpain 2–deficient cells demonstrate that GFP-cortactin persists at invadopodia for longer durations in the absence of calpain 2. Arrows indicate representative GFP-cortactin containing invadopodia. Duration measurements and assembly and disassembly rates ± SEM are shown. Asterisks indicate statistical significance compared with control cells based on t test (P < 0.05). See Videos 7 and 8 (available at http://www.jcb.org/cgi/content/full/jcb.200708048/DC1). Bars, 10 μm.