Figure 3

EGF receptor tyrosine kinase and β-catenin are required for disc colony formation. A) Western blots of total and activated β-catenin. HCT-116 cells were grown in 3-D matrigel for 6 days in either E or RNEW media. Cells were then lysed and subjected to Western blot analysis to determine the levels of total and activated β-catenin as described in Materials and Methods. GAPDH was used as a loading control. B) Quantitation of round or flat colonies of β-catenin knockdown (β-cat KD) HCT-116 cells grown in E or RNEW media 6 days. Results are expressed as mean percent of round or disc morphology +/− standard error of the mean (SEM), n>3, *p < 0.01. C) Western blots of total and phospho-EGFR. HCT-116 cells were grown in 3-D matrigel for 6 days in the presence or absence of 50 nM gefitinib. Cells were then lysed and subjected to Western blot analysis to determine the levels of total and phospho-EGFR as described in Materials and Methods. GAPDH was used as a loading control. D) Quantitation of round or flat colonies of HCT-116 cells grown in E, RNW or RNEW media in the presence or absence of 50 nM gefitinib for 6 days. Results are expressed as mean percent of round or disc morphology +/− standard error of the mean (SEM), n>3. *=p<.01. E) Quantitation of luciferase activity of the BRE-reporter. BRE-luciferase and β-galactosidase reporters were co-transiently transfected into HCT-116 cells. 24 hours later, cells were treated with the indicated growth factors for an additional 24 hours and luciferase and β-galactosidase were measured. Values are normalized to β-galactosidase activity. Results expressed as the mean percent of normalized luciferase activity +/− SEM, n=3.