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BMP9 induction of mRNA transcription is Smad4-dependent. A and B, HPAECs were transfected with siSmad4 or siCP (15 nm each) using Dharmafect1 (DH1). After 28 h, cells were serum-restricted for 16 h, followed by treatment with BMP9 (1 ng/ml) in 0.1% for 8 h. Total RNA was extracted and cDNA prepared. The expression of Id1 and Id2 (A) and IL-8 and E-selectin (B) were determined by qPCR, with expression being normalized to β-actin and expressed as the fold-change relative to 0.1%. Data are presented as the mean ± S.E. of five experiments. C, the expression of Smad4 was determined using qPCR, confirming selectivity. Data are presented as the mean ± S.E. of five experiments. D, cell lysates were immunoblotted for Smad4 to confirm the specific loss of receptor expression. *, p < 0.05 compared with DH1 plus BMP9.

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