miR-204 modulates the Src–STAT3–NFATc2 pathway in PASMCs from a patient with PAH (PAH-PASMCs). (A) miR-204 regulates human PASMC apoptosis and proliferation. Analysis of PASMC proliferation (PCNA nuclear localization) and serum starvation–induced apoptosis (TUNEL staining) of PASMCs from PAH patients (n = 3 patients) and healthy controls (n = 5 individuals). Control (Ctrl) or miR-204 antagomirs (Inh) were added as indicated. (B and C) miR-204 down-regulation in PAH-PASMCs increases Src and STAT3 activation. Total and phosphorylated Src (B) and total and phosphorylated STAT3 (C) in PASMCs from three PAH and five control patients monitored by Western blots are shown. Control or miR-204 antagomirs or mimics were added when indicated. Smooth muscle actin (SM-actin) was used as a loading control. Representative Western blots are shown. (D) miR-204 down-regulation in PAH-PASMCs increases NFAT expression and activation in human PASMCs. NFATc2 mRNA expression (left) and activity (right) were measured by qRT-PCR (left) and luciferase assay (right) in PASMCs from control or PAH patients treated either with control or miR-204 antagomir. Error bars represent mean value ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).