BMP9-dependent SDF1 induction is coordinated with CXCR4 repression. (A) RT-PCR (top panel) and quantitative RT-PCR (bottom panel) of HUVEC cDNA after ALK1 shRNA treatment. Bars represent the mean ± SEM; n = 3 experiments. (B) shRNA suppression of ALK1 inhibits the BMP9-dependent induction of SDF1 mRNA. (C) ELISA for CXCL12/SDF1 protein secreted into the conditioned medium of untreated control and BMP9-treated HUVECs, showing the effect of knockdown of endoglin and ALK1. Bars represent the mean ± SE for 3 experiments. (D-F) BMP9 repression of CXCR4 receptor mRNA expression. (D) RT-PCR indicated that the SDF1 chemokine receptor, CXCR4, was repressed by BMP9 in primary endothelial cells; shown are HAECs and HMVEC-C. (E-F) Quantitative RT-PCR indicating that BMP9 (5 ng/mL, 24 hours) repression of CXCR4 mRNA HUVECs requires endoglin and ALK1. BMP9-dependent repression was relieved after shRNA suppression of either endoglin (E; P = .012) or ALK1 (F; P ∼ .033).