Fig. 1.

Bone morphogenetic protein type II receptor (BMPR2) knockdown in human pulmonary endothelial cells (HPAECs) results in a dysfunctional endothelial cell phenotype. A: BMPR2 mRNA levels were assessed 48 h after BMPR2 knockdown. BMPR2 mRNA was measured in four different donors and is presented as geometric means ± SE. B: BMPR2 protein levels were examined after 48 h to assess the efficiency of gene silencing. For BMPR2 protein, densitometric quantification (means ± SE) relative to β-actin and normalized to the siControl is shown for three different donors; the inset shows a representative Western blot. ATP (C) and MTS (D) assays were used to quantify cell proliferation. Forty-eight hours after BMPR2 knockdown, HPAECs were transferred to 96-well plates for 6 h and serum starved for 24 h, before proliferation was assessed for 72 h in complete media. Data (means ± SE) represent five independent experiments for ATP assay and four independent experiments for formazan assay using different donors. E: representative images of cell migration measured in Oris 96-well plates. F: percent closure (means ± SE) of monolayer defects after 36 h of incubation for three different donors is shown. Cytoskeletal architecture examined by confocal microscopy with costaining for BMPR2 and phalloidin (F-actin) (G) or BMPR2 and paxillin (H). Cells were counterstained with DAPI. Confocal images are representative of results obtained from four different HPAEC donors. Scale bars: 10 μm. *P < 0.05; **P < 0.01.