Figure 4.

Rac1 is required for the chemotactic migration and invasive potential toward HGF. ASPC-1 and Panc-1/3D7 cells (3 x 10⁶) were electroporated only (Cont) or electroporated with 200 nM siRNA specific for Rac1 or a nontargeting control (Non) as indicated and analyzed 48 hours after siRNA treatment for chemotactic migration and invasion (A) toward medium/BSA or 50 ng/ml HGF; *P < .001 compared to Cont and Non. Immunoblot of cell lysates was used to confirm downregulation of Rac1 for the ASPC-1 cells (lower left) and Panc-1/3D7 cells (lower right). (B) Panc-1/3D7 cells (3 x 10⁶) were electroporated only (Cont), treated with 200 nM siRNA specific for the integrin subunits β4 and α6 or a nontargeting control (Non) as indicated. After 48 hours of siRNA treatment, cells (3 x 10⁶) were plated on laminin-1 and left untreated or treated for 5 minutes with 50 ng/ml HGF. Cell lysates were then analyzed for Rac1 activity using the PBD binding assay as described in the Materials and Methods section. (C) Densitometric analysis of three different exposures of immunoblots from (B) of activated (PAK-associated) Rac1. Data are reported as values for activated Rac1 divided by value for total cellular Rac1. *P < .001 compared to Cont and Non, BSA, and HGF. Inset shows immunoblot of integrin β4 to confirm target downregulation and actin as loading control. Data are representative of three independent experiments.