ROCK inhibition alters the cellular spatial distribution of MLC. (A) Representative micrographs of top (top) and mid sections (bottom) of cysts fixed at days 3 and 5 grown under no treatment (c3d and c5d) and treated with Y-27632 (Y3d and Y5d). Cysts were stained with DAPI and phalloidin. Bar, 10 µm. (B) We used GFP-MLC as an indicator for cortical tension (Yu et al., 2003). MDCK cells expressing GFP-MLC were cultured in Matrigel solution. Aphidicolin and Y-27632 were applied separately from day 0 or day 3. Cells were then fixed at day 5 and stained for PCX and nuclei. In control cysts, MLC is concentrated at the basal surface in an untreated cyst. When Y-27632 is applied from day 0, MLC is no longer accumulated at the basal surface, and instead is diffuse in the cytoplasm. In contrast, Aphidicolin does not change the localization pattern of MLC, which remains mainly at the basal surface. For a more detailed quantification of this effect see Fig. S5. Bar, 20 µm.