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. 2008 May;10(5):408–417. doi: 10.1593/neo.07868

Figure 5.

Figure 5

Tiam1 expression is required for Rac1 activity, migration, and invasion toward HGF of most pancreatic cancer cell lines that have high levels of integrin α6β4. (A) Pancreatic cancer cell lines were assessed for Tiam1 mRNA expression with real-time PCR. Data were normalized to 18S and reported relative to MiaPaCa2. (B) Lysates from the indicated cell lines were immunoblotted for Tiam1 (top panels) and tubulin as a loading control (bottom panels). (C) Panc-1/3D7 cells (3 x 106) were electroporated only (Cont), or electroporated with 200 nM siRNA that is nontargeting (Non) or specific for Tiam1, as indicated, and analyzed 48 hours after siRNA treatment for chemotactic migration on laminin-1 or collagen invasion toward 50 ng/ml of HGF; *P < .001 compared to Cont and Non-treated cells. Immunoblot of Tiam1 for siRNA target confirmation and actin as loading control (top panel). (D) Panc-1/3D7 cells were either treated with 200 nM siRNA specific for Tiam1 or with a nontargeting control (Non) as indicated. After 48 hours of siRNA treatment, cells were left untreated (-) or transfected with vector or a full-length Tiam1 construct. After 48 hours, cells were assessed for their ability to migrate toward HGF (bottom panel) as in (C). Immunoblot analysis demonstrates Tiam1 levels after Tiam1 transfection and siRNA ablation with actin as loading control (top panel). *P < .001 compared to Non/vector and Non/Tiam1 FL; ‡P = .001 compared to Tiam1/vector. (E) Panc-1/3D7 cells from (C) were assessed for Rac1 activity as performed in Figure 4C. Graph in (E) shows densitometry analysis from two different blot exposures; *P < .001 compared to Cont/BSA; †P = .003 compared to Non/BSA; ‡P < .001 compared to Cont and Non/HGF. Data are representative of three independent experiments. (F) Panc-1/3D7 cells (3 x 106) were electroporated only (Cont), treated with 200 nM siRNA specific for the integrin subunits β4 or α6, alone or in combination, as indicated. After 48 hours of siRNA treatment, cells (3 x 106) were lysed and probed for the presence of Tiam1, integrin β4, and actin.