FIGURE 5.

Combination of gefitinib with cilengitide shows enhanced inhibitory effects on TGF-β1-induced EMT expression and Smad signaling. A549 cells were treated with gefitinib (1 µM) and cilengitide (3 µM) individually or in combination, and then incubated with TGF-β1 (5 ng/mL) for 72 h (A and B) or 48 h (C) or 24 h (D and E). The expression of EMT markers (A) and phosphorylation of Smad2/3 (C) were measured by western blot analysis. Actin was used as a loading control. (B) The expression of vimentin was measured by immunocytochemistry. Nuclei were counterstained with DAPI. Scale bars = 100 µm. (D and E) After RNA extraction and cDNA synthesis, we performed qRT-PCR to measure the expression of CDH2 and VIM mRNA using GAPDH as an internal control. ^# p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. the group treated with TGF-β1 only.