GA inhibits GTP binding and the activation of downstream effectors. (A) GA dose-dependently inhibits GTP binding and the activation of downstream effectors in vitro. (B) and (C) Western blots showing the levels of activated downstream effectors, c-RAF phosphorylated at S338 and S259 and phosphorylated ERK1/2 levels, in A549 cells and HepG2 cells after GA treatment. GAPDH expression was used as an internal control for normalization. The assay was performed three times, Error bars indicate means ± S.D. (*P < 0.05, **P < 0.01, ***P < 0.001 compared to the control). (D) and (E) A549 cells were cultured with 20 or 40 µmol/L GA. Migration (upper panel) and invasion (lower panel) were investigated using Transwell and Matrigel assays (n = 5), scale bar: 50 µm. (F) and (G) PI staining was performed to investigate the effect of GA on the cell cycle. Cells were treated with GA (20 or 40 µmol/L) for 24 h and then stained with PI. GA induced S phase arrest in A549 cells. Histogram shows the percentage of cells in G0/G1, G2/M and S Phase (n = 3).