SK OV 3 was tested on three sep arate occasions to ensure consistency of results. Each of the samples was Bosutinib mechanism plated at 20,000 cells well into one well of two separate Greiner 24 well culture plates. Both plates were main tained under normoxic conditions for 48 hours Inhibitors,Modulators,Libraries to allow for cell adherence and equilibration. After 48 hours, one plate remained in normoxic condi tions while the other plate was transferred to a NAPCO Series 8000WJ Water Jacketed CO2 Incubator where hypoxic condi tions were established. Nitrogen gas was injected to purge the incubator of oxygen resulting in a final O2 concentra tion of 1% while the CO2 concentration was maintained at 5%, as described by Mukherjee et al. Plates were incubated for an additional 48 hours.

At the end of the incubation period, Inhibitors,Modulators,Libraries the confluency for Inhibitors,Modulators,Libraries each sample was recorded and the supernatant was collected and stored at 80 C. Confluency is the percentage of substrate with adherent cell growth, determined subjectively by a trained technician. ELISA Collected supernatants were sent to Millipore Corpora tion for protein evaluation via the Bead lyte CytokineProfiler Testing Service, an ELISA based assay. Evaluated angiogenesis related cytokines and growth factors included VEGF, PDGF AA, PDGF AA BB, IL 8, bFGF, EGF, IP 10, Flt 3 ligand, TGF 1, TGF 2, and TGF 3. Additionally, RANTES, an analyte not related to angiogenesis, was tested as a negative con trol for a subset of samples. For each analyte, two rep licates were performed using 40 l of supernatant per replicate.

Statistical analysis For Inhibitors,Modulators,Libraries each analyte, protein expression levels in the nor moxic and hypoxic conditions of all samples were com bined into a scatter plot. Then, a linear regression of the curve fit for protein concentration under the hypoxic ver sus normoxic condition was generated for each analyte tested. For all linear regressions, y mx b, y is the concen tration produced in the hypoxic environment and x is the concentration produced in the normoxic condition. From this regression, the slope, intercept, and correlation of determination were calculated. The strength of each linear relationship was determined by the r2 value of the tem, and 1 unknown primary. Additionally, five cell line samples were tested including A549, one sample. MDA MB 231, one sample. and SK OV 3, three samples. All samples were evaluated under both normoxic and hypoxic environments in parallel.

A strong linear relation ship for the Inhibitors,Modulators,Libraries confluency of the normoxic versus hypoxic condition existed across all samples, with a linear regres sion of y 0. 9917x 1. 516. Hypoxia induced expression of angiogenesis related factors Moderate to strong Abiraterone mw linear relationships of the protein expression levels between hypoxic and normoxic condi tions were observed in eight of the eleven angiogenesis related factors analyzed.