BMP9 treatment of endothelial cells results in a prominent chemokine response. (A) HMVEC-D were transfected with BRE-Luciferase reporter construct and then treated with increasing doses of recombinant BMP9 for 24 hours. Cells were collected and analyzed for Luciferase activity. Data were normalized to the pGL3 empty Luciferase reporter. Data are mean ± SEM. Endothelial cells responded to physiologically relevant concentrations of recombinant BMP9¹⁰  with dose-responsive increases in BRE-Luciferase promoter activity (left panel), which is confirmed by quantitative RT-PCR for BMP-responsive ID1 mRNA (right panel). (B) HMVEC-D were treated with 5 ng/mL BMP9 (+) or control (−), and total RNA and cell lysates were collected for analysis. RT-PCR (left panel, 4 or 24 hours after BMP9 treatment) and Western blotting (right panel, 24 hours after BMP9 treatment) show increased endoglin transcript and protein levels in response to BMP9 stimulation. (C) Cell component, Gene Ontology for biologic processes, and molecular function (x-ray/ANOVA) analyses suggest that BMP9 stimulation predominantly results in an alteration in endothelial cell extracellular protein expression and adhesion (upper left profile), chemokine receptor (right panel), and chemotaxis/inflammatory responses (lower left profile). Asterisks indicate emphasized extracellular, chemokine-related, and inflammatory pathways. (D) RT-PCR confirmation of the BMP9 down-regulated gene, E selection. Inset: Independent duplicate control untreated and triplicate cDNA preparations treated with 5 ng/mL BMP9. Quantitative real-time RT-PCR measurements indicate that BMP9 (5 ng/mL) produces 7- and 18-fold increases in endoglin and SDF1 mRNA expression at 24 hours after treatment, respectively. Bars represent the mean ± SE for 3 experiments.