Depletion of ALK1 in HAECs results in loss of correctly coordinated expression of VEGF. HAECs were transfected with scrambled (A) or ALK1 siRNA (B). The following day, the cells were treated with BMP-9 (10 ng/mL) for up to 24 hours (top); BMP-4 (40 ng/mL) in combination with BMP-9 inhibitor ALK1/Fc (200 ng/mL) for 12 hours, followed by no treatment for up to 24 hours (middle); and BMP-4 and ALK1/Fc for 12 hours, followed by BMP-9 for up to 24 hours (bottom). Cell samples were collected at the indicated time points (0-24 hours), and expression of ALK1, MGP, ALK5, and VEGF was determined by real-time PCR. The starting values from control cells (0S, −12S, and 40S) were used to compare expression also in the ALK1-depleted cells to facilitate comparisons between control and ALK1-deficient cells.