Induction of Myocd by TGF-β is Smad-dependent. A, hPASMCs were transfected with si-Ctr or siRNA against Smad4 (si-Smad4). After 24 h, cells were starved overnight followed by treatment with TGF-β for an additional 24 h. qRT-PCR analysis of Myocd, pri-miR-143/145, KLF4, or Smad4 mRNA, normalized to GAPDH, was performed (*, p < 0.05). B, hPASMCs were transfected with si-Ctr or si-Smad4. After 24 h, cells were starved overnight and then treated with TGF-β for 6 or 24 h, followed by immunoblot analysis of Myocd, Smad4, KLF4, or contractile markers (SMA and CNN). GAPDH serves as a loading control. C, upper, schematic of the Myocd promoter and location of SBE sites. Lower, PASMC were starved overnight and then stimulated with 400 pm TGF-β for 2 h. Cells were harvested, and ChIP assay was performed using anti-Smad2/3 or anti-Smad4 antibody. ChIP with nonspecific control IgG and anti-RNA polymerase II (RNA pol II) antibody were used as negative and positive controls, respectively. qRT-PCR was performed using different primer sets (A–C) to measure enrichment of the DNA fragment containing different regions of the Myocd promoter. Results were plotted as relative enrichment to input, mean ± S.E., n = 3. D, upper, schematic of Myocd promoter luciferase constructs. The B and C regions correspond to ChIP primers in C and SBE sites are numbered as in C. Lower, PAC1 cells were transfected with empty luciferase vector or the indicated Myocd promoter luciferase: pSBE1–7-luc, pSBE1–4-luc, pSBE3/4-luc, or pSBE6/7-luc. 24 h after transfection, cells were treated with TGF-β in starvation media for 24 h and subjected to luciferase assay. Results were plotted as relative luciferase activity normalized to β-galactosidase activity mean ± S.E., n = 3. *, p < 0.001. IP, immunoprecipitation.