Figure 4

RNEW activates ABL and AKT which are required for disc colony formation. A) Western blots of total and phospho-ABL. HCT-116 cells were grown in 3-D matrigel for 6 days in the presence or absence of 1μM imatinib. Cells were then lysed and subjected to Western blot analysis to determine the levels of total and phospho-ABL as described in Materials and Methods. GAPDH was used as a loading control. B) Western blots of total and phospho-AKT. HCT-116 cells were grown in 3-D matrigel for 6 days in the presence or absence of 50nM MK-2206. Cells were then lysed and subjected to Western blot analysis to determine the levels of total and phospho-AKT as described in Materials and Methods. GAPDH was used as a loading control. C) Quantitation of round or flat colonies of HCT-116 cells grown in E or RNEW media in the presence of vehicle, 1 μM imatinib, and 50 nM MK-2206 for 6 days. Results are expressed as mean percent of round or disc morphology +/− standard error of the mean (SEM), n>3, *p<0.001.