Effects of p34/ARPC2 or Nap1 depletion on collective cell migration. (A and B) p34/ARPC2 (A) or Nap1 (B) was depleted using siRNAs, followed by coimmunostaining for the indicated molecules. Yellow brackets indicate a region of cell junction in which the siRNA-targeted molecule is undetectable in immunostaining. White brackets indicate a region in which p34/ARPC2 or the Nap1 partner Abi is still faintly visible. Arrows point to LCs that are identified by E-cadherin distribution, and broken lines trace the basal margins of these LCs. Marginal and submarginal cells are shown. (C) Areas of E-cadherin–positive LCs in marginal and submarginal cells. The entire area of LC expanding below from each bilateral AJ was measured and compared. 31 junctions in control cells and 23 junctions in Nap1 or p34/ARPC2 siRNA-treated cells were analyzed. ****, P < 0.0001 (t test, one sided). (D) Migration distance of the marginal cells during 24 h in wound healing cultures. Values were obtained using four and six independent areas for control and Nap1- or p34/ARPC2 siRNA-treated cells, respectively. ****, P < 0.0001 (t test, one sided). (E andF) Sheets of wild-type Caco2 cells that were mixed in a 1-to-1 ratio with those expressing LifeAct-RFP, in which Nap1 or p34 p34/ARPC2 has been depleted with siRNAs. Magenta, LifeAct-RFP; green, actin. Photographed 48 h after wounding. In F, the ratio of siRNA-treated (LifeAct-RFP–labeled) cells to total cells in the three tandem zones of a culture was measured. Zones I, II, and III roughly correspond to a row of marginal cells, rows of submarginal cells, and an anterior group of interior cells, which span 58, 188, and 188 µm in width, respectively. We analyzed 11, 13, and 5 images for control and Nap1- and p34 p34/ARPC2 siRNA-treated cultures, respectively. Error bars represent SEM. Scale bars, 10 µm in A and B; 100 µm in E.