Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.