In vitro buy inhibitor cell proliferation assays Cells were seeded in 96 well plates using DMEM High Glucose medium supplemented with 10% FBS and 2 mM L glutamine . or DMEM High Glucose Inhibitors,Modulators,Libraries medium supplemented with 2 mM L glutamine and 50 ng/mL of recombinant human VEGF. Cells were cul tured overnight before being treated, in duplicate, with 10 point serial Inhibitors,Modulators,Libraries dilutions of single agent motesanib or docetaxel for 72 hours at 37 C. Cell viability was mea sured using an ATPlite 1 step luminescence assay as described previously. To assess the effect of motesanib plus chemo therapy combination treatment on in vitro proliferation, A549, Calu 6, NCI H358, NCI H1299, and NCI H1650 cells were seeded as described above and then treated with motesanib plus serial dilutions of cisplatin or docetaxel in PBS for 72 hours at 37 C.

Cell viability was determined using the ATPlite luminescence assay as described. In vitro tumor cell VEGFR2 phosphorylation Phosphorylation of VEGFR2 in tumor cells and HUVECs was assessed as described. Briefly, Inhibitors,Modulators,Libraries HUVECs, A549, Calu 6, NCI H358, NCI H1299, and NCI H1650 cells were cultured in full serum condi tions, serum starved conditions, and serum starved conditions plus recombinant human VEGF at a final concentration of 50 ng/mL for 5 minutes before harvest ing. Cells were lysed, and VEGFR2 protein was immuno precipitated using an anti human VEGFR2 polyclonal antibody and Protein A beads. Phos phorylated VEGFR2 protein was detected by Western blot using 4G10 horseradish peroxidase linked antiphosphotyrosine monoclonal antibody.

To detect total VEGFR2, the blot was stripped and reprobed with the polyclonal anti VEGFR2 antibody. Signals were detected with chemoluminescence using SuperSignal West Pico. Blot imaging was performed with a VersaDoc Imaging System Model 500 and blot quantification with ImageQuant 5. 2 software. Tumor xenograft models Animals were obtained from the following sources fe male Inhibitors,Modulators,Libraries CD1 nu/nu mice from Charles River Laboratories, female athymic nude mice from Harlan Sprague Dawley, and CB 17 severe combined immunodeficiency mice from Charles River Laboratories. Procedures met the standards of the Amgen Animal Care and Use Committee. The facilities where experiments involving animals were conducted were approved by the Association for Assessment and Ac creditation of Laboratory Animal Care. On day 0, mice were injected subcutaneously on the right flank with either Inhibitors,Modulators,Libraries of the following cultured Calu 6 . A549 . NCI H358 . NCI H1299 . or NCI H1650 cells. After tumors became established, mice received the following agents either alone or in combination as specified by the experimental protocols vehicle or motesanib orally once daily or twice daily Nutlin-3a clinical . PBS or intraperitoneal cisplatin once weekly . or PBS or intraperitoneal docetaxel.