Figure 3: Altered BMP6 responses in HPAH PASMC are mediated via ALK2 and ACTR-IIA.

(a,b) Proliferation of human control (a) and HPAH (b) dPASMCs following 6 days treatment every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) in the presence of LDN193189 (250 nM) (n=3 control and HPAH cell lines). (c,d) Proliferation of human control (c) or HPAH (d) dPASMCs on day 6 following transfection with DharmaFECT1^TM alone (DH1), siALK2 or non-targeting siRNA control (siCP) and treatment every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) as indicated (n=3 control and HPAH cell lines). (e,f) Human HPAH dPASMCs following transfection with DH1 alone, siACVR2A or siCP and treated with TNFα (1 ng ml⁻¹) and/or BMP2 (10ng ml⁻¹) or BMP6 (10 ng ml⁻¹) for 24 h as indicated. (e) Representative immunoblots of phospho-Smad1/5, total Smad1 and ID1 expression, reprobed for α-tubulin to ensure equal loading. The data shown are representative of three HPAH cell lines. (f) ID1 mRNA expression normalized to ACTB (n=3). (g) Proliferation of human HPAH dPASMCs on day 6 following transfection with DH1 alone, siACVR2A or siCP. Cells were treated every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) as indicated (n=3 HPAH cell lines). One-way analysis of variance with post hoc Tukey’s for multiple comparisons used in a,b,c,d,f and g. *P≤0.05, **P≤0.01, ***P≤0.001. Error bars and mean±s.e.m.