DAAM1 depletion promotes protrusion of cell edges. (A) Live imaging of membrane-EGFP introduced into shControl or shDAAM1 cells in confluent cultures. Cells were transfected with membrane-EGFP, leaving the majority of them untransfected, and cultured for 1 d on collagen gels. Representative still images were chosen from Video 5 (for shControl) and Video 6 (for shDAAM1). The labeled cell is surrounded with nonlabeled cells. Arrows indicate representative protrusions. The results obtained using the shDAAM1-2 line are shown. (B) Quantification of the frequency of protrusion per 5 min in two independent lines of shDAAM1 cells or an shControl line. 6–21 cells were examined for each experiment. (C) Mixed cultures of shControl or shDAAM1 cells. Cells were labeled with CMTPX and mixed with nonlabeled cells to culture on collagen gels for 2 d. shDAAM1 cells form longer and more protrusions than shControl cells, especially when mixed with control cells. Arrows indicate representative protrusions. The results obtained using the shDAAM1-2 line are shown. (A and C) Bars, 10 µm. (D) Quantification of protrusion number and length in CMTPX-labeled cells as indicated in Materials and methods. (B and D) Histograms represent the mean of three experiments. Error bars indicate SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.