De-activated EGFR constitutes the main component of the vacuole-like structure in ACCS-AS: Cells were double-stained with anti-EGFR (green) and anti-phosphorylated EGFR(red). Immunocytochemistry showed that vacuole-like structure formed in ACCS-AS cells upon gefitinib treatment contained mostly de-activated EGFR. Attached cells were markedly reduced at 6 hours. In ACCS cells, vacuole-like formations were not detected. At 6 hours, the number of attached cells was also reduced. In ACCS cells, there were some evidences of internalization of EGFR to the cytoplasm.