Inhibition of EGFR suppresses SP frequency and self-renewal. (A) H1650 and A549 cells revealed a significant decrease in SP frequency after treatment with 10 μg/ml EGFR-neutralizing antibodies for 5 days. (B) Frequency of SP cells in control antibody (1) and EGFR-neutralizing antibodies (2) treated cells. (C) Western blot analysis for ABCG2 and phospho-EGFR after EGFR-neutralizing antibody treatment. (D) SP frequency in NSCLC cells transfected with siRNA against EGFR or a control siRNA. (E) Expression of ABCG2 and EGFR in antibody treated cells. (F and G) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated per well 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.