Each point was measured in duplicate, and each experiment was replicated at least 4 times. Calculation animal study of affinity was performed by determining the IC50 using the curve-fitting program Prism GraphPad 4.0 (GraphPad Software, Inc., La Jolla, CA). Measurement of [3H]Inositol Phosphates. [3H]Inositol phosphates (IP) were measured in the different cells as described previously elsewhere (Rowley et al., 1990; Benya et al., 1992, 1994). In brief, all cells except N417 were subcultured into 24-well plates in regular propagation media and then were incubated for 24 hours at 37��C in a 5% CO2 atmosphere: hGRP-R (0.15 �� 106 cells/well), hNMB-R (0.03 �� 106), hBRS-3 (5 �� 105), HuTu-80 (0.25 �� 106), and NCI-H1299 (1 �� 106). The cells were then incubated with 3 ��Ci/ml myo-[2-3H]inositol in growth media supplemented with 2% FBS for an additional 24 hours.

After the incubation, the 24-well plates were washed by incubating for 30 minutes at 37��C with 1 ml/well PBS (pH 7.0) containing 20 mM lithium chloride. The wash buffer was aspirated and replaced with 500 ��l of IP assay buffer containing 135 mM sodium chloride, 20 mM HEPES, pH 7.4, 2 mM calcium chloride, 1.2 mM magnesium sulfate, 1 mM EGTA, 20 mM lithium chloride, 11.1 mM glucose, and 0.05% BSA (w/v), and was incubated without (control) or with different concentrations of the peptides studied. The N417 cells (1 �� 106 cells/ml), which grow in suspension, were centrifuged to remove the RPMI 1640 medium and were incubated directly with myo-[2-3H]inositol in RPMI 1640 medium with 2% of FBS for 24 hours (Ryan et al.

, 1998b; Sancho et al., 2010). The N417 cells were centrifuged to remove the wash buffer, and the cells were distributed in 5-ml tubes which were incubated with peptides in 500 ��l of IP assay buffer. After 60 minutes of incubation at 37��C, the experiments were terminated by the addition of 1 ml of ice-cold 1% (v/v) hydrochloric acid in methanol. The total [3H]IP was isolated by anion exchange chromatography as described previously elsewhere (Rowley et al., 1990; Benya et al., 1995; Ryan et al., 1998a; Uehara et al., 2011). Samples were loaded onto Dowex AG1-X8 anion exchange resin columns, washed with 5 ml of distilled water to remove free [3H]IP and then washed with 2 ml of 5 mM disodium tetraborate/60 mM sodium formate solution to remove [3H]glycerophosphorylinositol; after this, 2 ml of 1 mM ammonium formate/100 mM formic acid solution were added to the columns to elute the total [3H]IP.

Each eluate was mixed with scintillation cocktail and measured for radioactivity in a scintillation counter. Western Blot Analysis. Western blot analysis Cilengitide of the various cells was performed as previously described elsewhere (Berna et al., 2007; Sancho et al., 2010). Balb/hBRS-3 and NCI-N417 cells were washed with PBS and incubated with starvation medium (DMEM or RPMI 1640 medium without FBS) for 2 hours and 3 hours, respectively, at 37��C in a 5% CO2 atmosphere.