Cell a version 3.1

Treated cells were fixed and permeabilized with 0.1% Triton x-100 for 5 min at room temperature and then incubated with Alexa Fluor 488- labeled Phalloidin (A12379, Molecular Probs, Inc, OR, USA) and PE-conjugated αvβ3 antibody (LM609, Chemicon International). Hoechst 33342 was used for nuclear staining (Sigma-Aldrich). Cells were visualized by a fluorescent microscopy equipped with a camera (Model IX71, Olympus, Hamburg, Germany) with a 20X/0.50 objective lens and Cell^A (Version 3.1) Olympus software imaging.

CAG cells were seeded (100,000 cells/96-well plate) and starved for 48 h in serum-free media. Each well was scratched in the middle and T3 (1 nM) or T4 (100 nM) were added for 0, 24, 48, 72 and 96 h. Cells were visualized by light and florescent microscopy equipped with a camera (Model IX71, Olympus, Hamburg, Germany) with a 20X/0.50 objective lens and Cell^A (Version 3.1) Olympus software imaging at each time point. After 96 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton x-100 for 5 min. 1% BSA was used for blocking, followed by Hoechst and Phalloidin staining.

For adhesion assays 96-well plates were pre-coated for 30 minutes with fibronectin (15 μg/mL), RGD (2.5 μg/mL) or BSA (10 μg/mL), washed twice with PBS and blocked for 30 minutes in BSA (2mg/mL). CAG cells were seeded (100,000 cells/24-well plate) under serum-free conditions for 24 hours before the addition of T3 (1 nM) or T4 (100 nM) for an overnight incubation. Afterwards cells were collected, counted and an equal number of cells were re-seeded for 30 minutes in 37º C in the pre-coated plates (50,000/96-well plate). Unattached cells were washed twice with PBS and adhered cells were stained with Hoechst 33342. Cells were visualized by a florescent microscopy equipped with a camera (Model IX71, Olympus, Hamburg, Germany) with a 20X/0.50 objective lens and Cell^A (Version 3.1) Olympus software imaging.