In vitro activity: ATN-161 treatment up to 100 μmol/L shows no significant effect on tumor cell proliferation compared with the vehicle-treated control group of cells. However, ATN-161 significantly inhibits MAPK phosphorylation with maximal effects observed at 20 μmol/L of ATN-161 after 30 minutes of treatment.

Kinase Assay: Binding to purified integrins and localization of ATN-453 to a particular integrin subunit was carried out as follows. α5β1-integrin (Chemicon; 0.2 μmol/L), ATN-453 (10 μmol/L), and 2 mmol/L Mn2+ in 50 mmol/L HBS were incubated at room temperature for 2 h Samples were then resolved under nonreducing conditions on a 4% to 12% NuPAGE Bis-Tris gel, then transferred to a polyvinylidene difluoride membrane by Western blot, and probed with Streptavidin–horseradish peroxidase to identify the ATN-453–bound band. Separate blots were also probed with either an anti-α5, an anti-β1, or an anti-β3 antibody followed by the appropriate secondary antibody to identify the subunit that interacts with ATN-453. Alternatively, purified α5β1 was incubated with anti-β1 antibodies followed by incubation with ATN-453, as previously described. Under nonreducing conditions, the α5subunit runs at ~160 kDa and β1 at ~110 kDa.

Cell Assay:MDA-MB-231 (1 × 10⁶) cells are plated in 100 mm Petri dishes for 24 hours, then serum-starved overnight before treatment with vehicle or ATN-161 (1-100 μmol/L) for different time periods (15-60 minutes). Western blot analyses are carried out using antibodies against focal adhesion kinase, phosphorylated FAK, mitogen-activated protein kinase (MAPK), phosphorylated MAPK, and β-tubulin as previously described. Western blots are detected using enhanced chemiluminescence detection reagents.