Human ProS inhibits VEGF-A–induced EC proliferation, migration, and signaling. (A) The percentage of BrdU incorporation induced by the indicated factors. ECs were seeded at a density of 6 × 10³ cells/well in 96-well plates in growth factors containing medium for 24 hours, switched to a growth factor-depleted medium containing the indicated factors at the specified concentrations. BrdU incorporation was measured by ELISA. Data obtained from 3 independent experiments each in triplicates are expressed as percentages of control ± SD. ***P < .001. **P < .01. ns indicates not significant. (B-C) Western blot analysis of MAPK-Erk1/2 activation after cultured EC exposure to the indicated factors. Subconfluent EC cultures were exposed in a growth factor–depleted medium to ProS (10 μg/mL) or vehicle for 15 minutes and then stimulated with VEGF-A (20 ng/mL) for 5 minutes. Cell cultures were then lysed, and equivalent amounts of protein form each sample were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with either antiphosphorylated Erk 1/2 or anti–Erk 1/2 antibodies. Band intensities were quantified and are represented in panel C as a percentage of control of the ratio of phosphorylated Erk1/2 over Erk1/2 ± SD. Data were obtained from 4 independent experiments each in triplicates are expressed as percentages of control ± SD. ***P < .0001. *P < .05. (D) The number of migrated ECs induced by the indicated factors. A total of 2.5 × 10⁴ ECs were suspended in culture medium containing 0.25% BSA, seeded in the upper compartment, and separated from the lower compartment by a 5-μm pore size polycarbonate filter coated on both sides with 0.1% gelatin. The lower compartment contained the factor under study at the indicated concentrations diluted in 0.5 mL culture medium. After 4 hours of incubation, the upper surface of the filter was scraped, and cells present in the lower compartment were fixed, stained, migrated cells were photographed under the microscope, and counted on 15 fields using ImageJ Version 1.39o software. Data were obtained from 3 independent experiments, each in triplicates, expressed as percentages of control ± SD. ***P < .0001. (E-F) Western blot analysis of Akt phosphorylation on Ser⁴⁷³ after cultured EC exposure to the indicated factors. Cell cultures were then lysed, and equivalent amounts of proteins from each sample were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with either anti-Akt or anti–phosopho-Ser⁴⁷³ Akt antibodies. Intensity of bands was quantified and are represented in panel F as a percentage of control of the ratio of phosphorylated Akt Ser⁴⁷³ over Akt ± SD. Data were obtained from 3 independent experiments, each in triplicates, expressed as percentages of control ± SD. **P < .01. *P < .05. ns indicates not significant.