K-Ras V12 and B-Raf V600E inhibit apical polarity establishment through ERK-mediated regulation of c-myc. (A and B) Single cells expressing control (pQCXIP GFP) or K-Ras V12 (pQCXIP GFP K-Ras V12) were embedded in matrix. After 2 d, cells were fixed and stained for aPKC, actin, and DNA (blue; A) and ZO-1, E-cadherin, and DNA (blue; B). Representative images are shown. (C) Two-cell structures expressing control (pQCXIP GFP), K-Ras V12 (pQCXIP GFP K-Ras V12), B-Raf V-600E (pQCFLAP B-Raf V600E), or c-myc (pBabe blast c-myc) were fixed and stained for aPKC and DNA. (bottom) K-Ras V12–expressing Caco-2 cells were plated as single cells in 3D and treated with 20 µM U0126. Cells stably expressing shmyc 2 were superinfected with K-Ras V12 (pQCXIP GFP K-Ras V12) retrovirus. 4 d later, cells were seeded into 3D culture. Cells were fixed 2 d later at the two-cell stage and stained for aPKC and DNA. Representative images are shown. (D–G) At least 50 two-cell structures were imaged for each condition in three independent experiments and analyzed for proper apical localization of aPKC. The means ± SEM are shown. **, P < 0.005; ***, P < 0.0005. Bars, 50 µm.