Figure 5

A PI3K inhibitor and MEK inhibitor inhibited migration and invasion abilities of SIP cell, iNP cell and iNP cell carrying EGFR (ERBB1) mutation. (A) The cells were cultured in 6-well plate, starved overnight in serum- and EGF-free FYAD medium, then incubated in the presence or absence of 20 µM LY294002 or 10µM U0126 for 48 h prior to harvesting cell lysates and analyzed by western blotting. The total and the phosphorylated levels of EGFR, AKT, and MAPK were detected by the antibodies described in “Methods” (left panel), and the intensities of individual bands were indicated by bar graphs (right panels). To expose one blot to several antibodies at the same time, the membrane was cut into several pieces at desired positions prior to exposure to specific antibodies. The grouping of blots in (A) was cropped from the same samples loaded in 2 gels with 7 blots. The original blots are presented in Supplementary Fig. S3. (B) Cell migration was investigated by wound healing assay as described in “Method” (left panel), and the wound area at 24 h after 20 µM LY294002 or 10 µM U0126 treatment were indicated by bar graphs (right panels). (C) Cell invasion was investigated by using transwell invasion assay as described in “Method” (left panel), and the number of invaded cells at 24 h after 20 µM LY294002 or 10 µM U0126 treatment were indicated by bar graphs (right panels). Note that treatment with 20 µM LY294002 and 10 µM U0126 significantly reduced migration and invasion abilities of the tested cells. “a, b, c” indicate a statistically significant difference (a: p < 0.05; b: p < 0.01; c: p < 0.001), and “ns” indicates not significant.