EGF activates Rac1 via the GEP100/Arf6 pathway that is required for EGF-induced cell migration.

<p>(A) Effect of EGF on the activation of Rac1. HepG2 cells were starved overnight, followed by treatment with 10 ng/mL EGF for the indicated times. Rac1 activation was determined as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038777#s4" target="_blank">Materials and methods</a>’. (B) Both GEP100-siRNA and GEP100-△PH transfection inhibit Rac1 activation. Cells transfected with GEP100-siRNA or GEP100-△PH were stimulated with EGF for 30 min, and Rac1 activation was examined. (C) EGF-induced activation of Rac1 was dependent on Arf6. Cells transfected with Arf6-T27N were stimulated with EGF for 30 min, and Rac1 activation was examined. (D) Effect of Rac1-T17N on EGF-stimulated cell migration. After transfection with the empty vector or Rac1-T17N, HepG2 cells were incubated with 10 ng/mL EGF for 8 h and the cell migration rate was determined by Transwell migration assay. (E-F) GEP100-△PH blocked EGF-stimulated membrane ruffling. Cells expressing empty vector or GEP100-△PH were stimulated with EGF for 15 min, fixed and stained for the distribution of actin using TRITC-conjugated phaolloidin (red). Cells were counterstained with DAPI (blue). Images are representative of at least 3 independent determinations. Magnification, ×400. Scale bar, 20 µm. *: <i>P</i><0.05, referring to the difference between cells treated with and without EGF. ^#: <i>P</i><0.05 (t-test), referring to the difference between cells transfected with Rac1-T17N, Arf6–T27N or GEP100 siRNA or GEP100-△PH plus EGF and the cells transfected with scrambled siRNA (irr siRNA) or empty vector plus EGF.