DAAM1 is important for epithelial integrity. (A) EpH4 cells stably expressing DAAM1-specific (shDAAM1-1 cells) or control (shControl cells) shRNA were cultured for 2 wk in Transwells and stained for the indicated molecules. (Top) Lateral views. Nuclei are mispositioned in DAAM1-depleted cells. (Bottom) Top views focused on an apical or basal focal plane. Abnormally large or small apical areas are detectable. Arrows indicate AJC positions. Bars, 10 µm. (B) Distribution of the apical area in shControl and shDAAM1 cells. Apical area was defined as the area encircled by ZO-1 immunostaining. 144 shControl and 80 shDAAM1 cells from three and four microscopic fields, respectively, were examined. (C) Rescue of the DAAM1 depletion phenotypes. shDAAM1 cells were additionally transfected with RNAi-resistant EGFP-DAAM1 plasmid and cultured for 2 wk in Transwells; the apical area variation was quantified. We examined 35–144 cells from three or four microscopic fields for each experiment. Histograms represent the mean of three experiments. Error bars indicate SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.