Transition from lamellipodia to blebbing in CK-666–treated HT1080 cells includes a filopodial stage. (A) Phalloidin staining in indicated conditions. (B) Time-lapse DIC imaging of an HT1080 cell exposed to increasing CK-666 concentrations. Time is in minutes:seconds relative to the first addition of CK-666 at 87.5 µM. The CK-666 concentration was increased to 120 µM at t = 24:22 and to 137 µM at t = 51:44. (C) Bleb dynamics from the boxed region in B at higher time resolution. Individual blebs are marked by arrows and arrowheads. CK-666 concentration during this time period was 120 µM. (D) Representative kymographs of bleb dynamics with (bottom) and without (top) a stationary phase. Arrows indicate time (t) and protrusion direction (d). Scale bars: 10 µm (A and B), 5 µm (C), 2 µm (D, d arrow), and 30 s (D, t arrow). See also Video 1. (E) Diagram illustrating a method to quantify distribution of protrusion. Numbers of blebs and filopodia were determined in each of the eight sectors of the cell perimeter. (F) Representative four graphs showing the numbers of blebs (Bl) or filopodia (Fp; y axis) versus the sector number (x axis). Pearson correlation coefficient (r) is shown for each graph. Upper left graph corresponds to the cell shown in B. (G) Distribution of individual r values (left) with mean ± SD (right) for n = 9 cells; P <0.0001 relative to r = 0 (two-tailed t test).