Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors for 4 days and western blot analysis was performed for the indicated proteins. (B) H1650-SPAdh cells were treated with MEK or PI3K inhibitor for 4 days and levels of Sox2 and activation of Akt or ERK was evaluated by western blot. (C) A549 and H1975 cells were treated with MEK or PI3K inhibitor for 5 days and the frequency of SP cells were examined by SP analysis. Data represents the fold change in mean (± SD) of SP frequency. (D) ABCG2 expression upon inhibitor treatment as detected by western blotting. (E) SP cell frequency in NSCLC cell lines transfected with specific siRNAs against Src or AKT. (F) ABCG2 expression in siRNA transfected cells as detected by western blotting. (G and H) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated from 1000 cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. * represent the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.