Evidence for a rapid endocytosis of ligand-induced pEGFR in the gefitinib-sensitive PC9 cells, but for an inefficient endocytic traffic of EGF-pEGFR in the gefitinib-resisitant QG56 cells. The PC9 (A, B) or QG56 (C, D) cells were incubated in the absence (A, C) or presence (B, D) of gefitinib at 37°C with Texas red-EGF for 5, 10, or 15 min, and cells were fixed and double-stained for pEGFR (green) as described in the Materials section. Superimposed images of pEGFR and Texas red-EGF are shown. The white arrowheads indicate the colocalization of the pEGFR-positive vesicles and Texas red-EGF-positive vesicular structures. It is notable that rapid endocytosis of EGF-EGFR occurs in PC9 cells, since large amounts of pEGFR-positive small vesicles co-stained with Texas red-EGF appear in the cytoplasm after 5 min incubation (a) and these co-stained vesicles are increased at 15 min (b, c). By contrast in QG 56 cells, pEGFR stainings are mostly associated with plasma membrane even after 15 min incubation (i). Further, gefitinib significantly suppresses phosphorylation of EGFR in NSCLC cell lines and amount of pEGFR stainings are considerably reduced during the incubation (d, e, f, j, k, l). Bar, 10 μm.