NRAS or CRAF amplification induces persistent activation of MAPK cascade in resistant cells. A. Heatmap of differential gene expression in PC9-v versus resistant cells. B. Western blot analysis of the MAPK pathway components in PC9-v and resistant cells in the presence or absence of erlotinib. C. Copy number analysis of NRAS and CRAF in PC9-v and resistant cells. *P<0.05, ANOVA followed by Tukey's post-test. D. PC9-v, ER1, ER2 and ER3 cells were treated with DMSO, erlotinib (1 μM), trametinib (0.5 μM) or erlotinib combined with trametinib (Combo). Phospho-EGFR and phospho-ERK were analyzed by western blot. E. Cell viability assay of PC9-v, ER1, ER2 and ER3 cell treated with DMSO, erlotinib (1 μM), trametinib (0.5 μM) or erlotinib combined with trametinib (Combo). *P<0.05, ANOVA followed by Tukey's post-test. F. PC9-v, ER1, ER2 and ER3 cells were treated as indicated and stained with crystal violet.