Figure 9.

Both p190 RhoGAP paralogs are required for normal epithelial architecture of MDCK cells propagated in Matrigel. (A) Outlines of median-size spheroids derived from MDCK cells with or without depletion of p190A and/or p190B cultured in Matrigel for 14 d. (B) Size (area) distribution of spheroids with or without p190A and/or p190B knockdown after 14 d of culture in Matrigel. The areas from ≥40 cysts from each condition were measured. (C) Immunolabeling for Scrib (green) and phalloidin staining to detect polymerized actin (red) in spheroids with or without p190A and/or p190B depletion after 10 d of culture in Matrigel. Nuclei were stained with DRAQ5 (blue). Bars: (low magnification) 10 µm; (inset) 2.5 µm. (D) Localization of YAP in MDCK cell spheroids with or without knockdown of p190A and/or p190B after 14 d of culture in Matrigel. The images for YAP (white) were superimposed on the DRAQ5 stain (blue) using the screen function in Photoshop. Optical sections close to the perimeter of spheroids are shown. Bars: (low magnification) 20 µm; (inset) 5 µm. (E) Staining of LATS2-KR–expressing MDCK cells cultured for 14 d in Matrigel with Alexa Fluor 594–phalloidin to detect polymerized actin (red) and DRAQ5 to label nuclei (red). LATS2 and ZsGreen are coexpressed from a bicistronic vector. Bar, 20 µm.