Figure 1. ErbB receptor inhibition restores aberrant morphogenesis of Caco-2 K-Ras^G12V cells in 3D culture.

A. Caco-2tet K-Ras^G12V cells were seeded into 3D cultures in the absence or presence of dox. Three days post seeding lumen expansion was induced by CTX. Cultures were fixed two days later and stained with DAPI (nuclei; blue) and phalloidin (F-actin; red). GFP is co-expressed with K-Ras^G12V (green). Shown are confocal sections of the midplane of representative cysts, nuclei are numbered in white (scale: 20 μm). B. The percentage of cysts with PSAL from (A) was determined (n>70; N=3). C. The number of nuclei in the midplane of cysts from (A) was counted (n=25; N=3). D. Caco-2tet K-Ras^G12V cells were seeded into 3D cultures. One day post seeding K-Ras^G12V-expression was induced by dox and cultures were left untreated or treated with 200 nM AZD8931. Two days later CTX was added. Cultures were fixed the next day and stained with DAPI (nuclei; blue) and phalloidin (F-actin; red). GFP is co-expressed with K-Ras^G12V (green). Shown are confocal sections of the midplane of representative cysts (scale: 20 μm). E. The percentage of cysts with PSAL from (D) was determined (n>60; N=3). F. The number of nuclei in the midplane of cysts from (D) was counted (n=25; N=3) G. Caco-2tet K-Ras^G12V cells were seeded into 3D cultures. One day post seeding K-Ras^G12V expression was induced by dox and cultures were left untreated or treated with 200 nM AZD8931. Lysates were generated four days after seeding and analyzed by immunoblotting using the indicated antibodies (pErbB2(Tyr1248)). Tubulin was detected as a loading control. A representative blot is shown (N=3).