Anchorage-independent growth of HME16C cell lines after EGFR inhibition. (A) Cell lines, as indicated, were grown in the soft agar assay for 21 days either in the presence of DMSO vehicle or 0.25 μM PD153035 EGFR inhibitor. Scale bar, 200 μm. (B) Number of colonies >100 μm in size for each cell line treated with either DMSO vehicle or 0.25 μM of the EGFR-specific inhibitor PD153035. Statistical analysis was performed using one-way analysis of variance with Bonferroni post-test analysis. NS, not significant, p > 0.05; *, p < 0.0001. (C) Activation of Erk after EGFR inhibition. Cell lines were grown in the presence of DMSO vehicle or 0.25 μM PD153035 for 48 hours, followed by western blotting for phosphorylated Erk (Phospho-Erk). The blot was stripped and probed with an anti-Erk 2 antibody (Erk 2) to verify equal protein loading. The phospho-ERK blot was overexposed in order to reveal minimal levels of protein.