Dysregulated AKT activity in Bmpr2 KD pulmonary vascular endothelial cells. (A) Characterization of SMAD-mediated signaling. Wild-type or Bmpr2 KD cells were treated with 100 ng/mL BMP4 or BMP9 for the indicated time. Cell lysates were immunoblotted with antibodies against phosphorylated or total SMADs. (B) Chemotactic migration of endothelial cells toward sphingosine-1-phosphate (S1P). Cells were pretreated with 200 ng/mL BMP4 or vehicle control. Data represent results from 3 independent experiments. (C) Effect of BMP signaling on AKT activation. Wild-type ECs were stimulated with S1P for the indicated time in the presence or absence of 200 ng/mL BMP4 or BMP9. Cell lysates were immunoblotted with phospho-AKT and total AKT antibodies. (D) Effect of BMP4 on S1P-, serum-, and VEGF-stimulated serine phosphorylation of AKT in wild-type versus Bmpr2 KD cells. The graph below the blot shows densitometry quantification of the phosphor-AKT levels normalized to total AKT. The open bars represent wild-type ECs whereas solid bars represent Bmpr2 KD cells. (E) Potentiation of EC apoptosis by BMP4. Cells were incubated in normal medium with 10% serum or serum-free medium (SFM) for 24 hours, and DNA fragmentation index was determined by Cell Death Detection enzyme-linked immunosorbent assay (Roche). Values are means plus or minus SD.