EGFR is overexpressed and activated in IR cells to promote IR cell invasion.

<p>(A) Quantitative analysis of EGFR mRNA levels from P and IR cells by qRT-PCR, represented as mean values ± S.D of relative mRNA level (***p<0.001) from 3 independent experiments, indicated as fold change relative to P cells. (B) Analysis of protein levels of total EGFR and phosphorylated EGFR (Tyr1068) by western blotting. Intensity of signals were quantified by densitometry and normalized with GAPDH. Results are represented as mean values ± S.D of relative protein level (**p<0.01) from 3 independent experiments, indicated as fold change relative to P cells. (C–E) Inhibition of EGFR activation induced a round morphology of IR cells. IR cells in 3D collagen gel were treated with 10 µM EGFR inhibitor PD168393 or DMSO as a control for 24 h. (C) Expression analysis of phosphorylated EGFR (Tyr1068) and total EGFR from PD168393- and DMSO-treated samples by western blotting. GAPDH was used as a control. (D) Representative images of cell morphologies of PD168393-treated samples versus the DMSO-treated control. (E) The percentage of elongated cells was quantified from 3 independent experiments including about 100 cells, ***p<0.001. (F) Quantification of speed in DMSO- or PD168393-treated IR cells in 3D collagen gel-sand for 6 h are represented as mean values ± S.D (***p<0.001) from 3 independent experiments including about 50 cells. (G) Confocal images of representative MFP488 phalloidin-stained IR spheroids in collagen gel for 0 h and IR spheroids treated with DMSO or PD168393 for 24 h. Scale bar, 200 µm. (H) Quantification of the area of spheroids by ImageJ software. (I) The aspect ratio of spheroids was calculated from perimeter²/[4π(area)]. Results are represented as mean values ± S.D (***p<0.001) from 3 independent experiments in triplicate.