Figure 2: TNFα alters BMP2 and BMP6 signalling dynamics and PASMC proliferation.

(a) BMPR2 mRNA expression, normalized to ACTB, in human dPASMCs from disease-free controls and HPAH patients stimulated with TNFα (1 ng ml⁻¹) for 24 h (n=3; Student’s t-test). (b,c) Pre-treatment of human dPASMCs from disease-free controls and HPAH patients with or without TNFα (1 ng ml⁻¹) for 23 h before 1 h stimulation with BMP2 (10 ng ml⁻¹) or BMP6 (10 ng ml⁻¹). (b) 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 control and HPAH cell lines. (c) ID1 mRNA expression, normalized to ACTB (n=3). (d) Proliferation of human dPASMCs from disease-free controls and HPAH patients after 6 days treatment. Cells treated every 48 h as indicated with BMP2 (10 ng ml⁻¹), BMP4 (10 ng ml⁻¹) or BMP6 (10, 25 or 50 ng ml⁻¹; n=3). (e) Proliferation of human dPASMCs from disease-free controls and HPAH patients after 6 days treatment. Cells treated every 48 h as indicated with TNFα (1 ng ml⁻¹) and/or BMP6 (10 or 50 ng ml⁻¹) (n=3 control and HPAH cell lines). (f) Proliferation of human control dPASMCs on day 6 after transfection with DharmaFECT1 alone (DH1), siBMPR2 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). (g) Proliferation of human HPAH dPASMCs on day 6 following transduction with adenovirus expressing full-length wild-type and kinase-dead D485G mutant BMPR-II cDNA and treatment every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) as indicated (n=3). One-way analysis of variance with post hoc Tukey’s for multiple comparisons used in (c–g). *P≤0.05, **P≤0.01, ***P≤0.001. Error bars represent mean±s.e.m.