Arf6 depletion dramatically alters cell morphology and reduces RhoB activity in response to HGF stimulation. (A) Western blot of Arf6 KD and rescue levels in HeLa cells with the indicated constructs. CTL, control. (B) Scanning electron micrographs (scale bar = 30 µm) and live-cell images (scale bar = 10 µm) of HeLa cells transfected with control or Arf6 siRNAs and rescued with Arf6-GFP or GFP-RhoB, stimulated with 0.5 nM of HGF (20 min). Arrows indicate the tail of cells. (C and D) Active GTP-RhoA (C) and RhoB (D) were examined in HeLa cell lysates of control and Arf6 KD. HeLa cells were pretreated for 2 h with DMSO or 10 nM of bafilomycin A1 (Baf) and stimulated with HGF at different time points as indicated. (E and F) Western blot showing RhoB (E) and RhoA (F) protein levels. All quantified data indicate mean values ± SEM from three independent experiments. P values based on comparisons with control: *, P < 0.05; **, P < 0.001.