TRAIL induces proliferation and migration of PASMCs. (A) TaqMan expression of TRAIL in explanted PASMC from patients with IPAH normalized using ΔΔCT with 18S rRNA as the endogenous control gene. Human PA-SMCs were serum starved for 48 h before stimulation with 1–100 ng/ml of recombinant TRAIL or 10 ng/ml PDGF-BB. (B) Proliferation was assessed by cell counting at 72 h. (C) migration was measured at 6 h using a Boyden Chamber assay and normalized relative to unstimulated cells (0.2% FCS). (D) Time course of p42/44 / ERK1/2 phosphorylation in PASMC using 30 ng/ml of TRAIL. Black lines indicate that intervening lanes have been spliced out. (E and F) Treatment of PASMC with the ERK1/2 inhibitor PD98059 inhibited TRAIL (30 ng/ml)-induced proliferation (E) and migration (F). (G) TaqMan expression of TRAIL receptors in explanted PASMC from patients with IPAH normalized using ΔΔCT with 18S rRNA as the endogenous control gene. Human PA-SMCs were stimulated with 30 ng/ml of recombinant TRAIL or 20 ng/ml PDGF-BB with blocking antibodies for TRAIL-R1, TRAIL-R3, or an IgG control. (H) Proliferation of human PA-SMCs in response to 30 ng/ml of recombinant TRAIL or 20 ng/ml PDGF-BB after preincubation with IgG or neutralizing antibodies for TRAIL-R1, TRAIL-R3, or an IgG control. Error bars represent mean ± SEM, and all experiments were performed in triplicate. For data using patient material (A and G), n = 3. All remaining figures, n = 5. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with 0.2% FCS, control, or 0 h samples.