WASH and exocyst complex are required for invadopodial degradation of the matrix. (A) Effect of WASH or Exo84 silencing on the density of MT1-MMPmCh–positive vesicles in the subplasma membrane region of MDA-MB-231 cells observed by TIRFM. Insets are the corresponding wide-field images showing similar MT1-MMP expression. Bars, 5 µm. (B) Box plots showing the density of subplasma membrane MT1-MMP vesicles. Number of cells analyzed from four experiments is indicated (n). *, P < 0.05; ***, P < 0.001 (compared with control cells treated with siNT). (C) MDA-MB-231 cells expressing MT1-MMPpHluorin and DsRed-cortactin were plated on cross-linked gelatin for 2–3 h and analyzed by dual color TIRFM. A merged image from a representative time-lapse series is shown. (D, top) Split signals from the boxed region in C showing accumulations of MT1-MMPpHluorin at cortactin-positive invadopodia. Bar, 5 µm. (bottom) Kymograph views of TIRF time series acquired every 10 s. (E) MDA-MB-231 cells expressing MT1-MMPpHluorin were treated with the indicated siRNAs, plated on cross-linked gelatin, and analyzed by TIRFM. Plots show the percentage of cells with MT1-MMPpHluorin–positive invadopodia. Values are means ± SEM from three independent experiments scoring a total of 150–200 cells for each cell population. ***, P < 0.001 (compared with cells treated with non-targeting siRNA).