Inhibition of cytohesin or Dock180 activity impairs HGF-stimulated Rac activation and wound healing. A,B) HGF-dependent Rac activation is impaired by SecinH3 or dominant negative Dock180. MDCK cells were incubated in the presence or absence of 20 ng/ml HGF for 6 hrs. SecinH3 (15 μM), a specific cytohesin inhibitor or adenovirus encoding the dominant negative Dock180 mutant, DockISP, were added as indicated. Rac-GTP was isolated by pulldown with GST-PBD. Pulldown samples and saved aliquots of the starting lysates were Western blotted with antibodies to Rac and Dock180. Four independent experiments were quantitated by densitometry (A) ** = p < 0.01, paired T test. Gels from one of these experiments are shown (B). C,D) SecinH3 and DockISP inhibit HGF-stimulated wound healing. MDCK cells were plated in 96 well Oris migration chambers (Platypus technologies) around plugs. 24 hours later the plugs were removed to create a wound. Wounded monolayers were incubated in the presence or absence of 1 ng/ml HGF with the addition of 30 μM SecinH3 or adenovirus encoding DockISP as indicated for 18 hours. Cells were then fixed and stained with crystal violet. The area of the remaining wound was measured using ImageJ. C) The extent of wound healing in multiple replicate samples from multiple independent experiments was quantitated: Control (n = 67 wells from 12 experiments); SecinH3 (n = 37 wells from 10 experiments); DockISP (n = 24 wells from 4 experiments). Data shown are mean ± standard error. ** = p < 0.01, T test. D) Representative images of the starting wound and wounds after 18 hours of migration in the presence or absence of HGF Bar = 500 μM.
Attar and Santy BMC Cell Biology 2013 14:9 doi:10.1186/1471-2121-14-9