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. Author manuscript; available in PMC: 2014 Sep 27.
Published in final edited form as: Circ Res. 2013 Jul 1;113(8):10.1161/CIRCRESAHA.113.301921. doi: 10.1161/CIRCRESAHA.113.301921

Figure 8. Progenitor-specific roles of Smad2 and Smad3 in regulating VSMC marker gene transcription and protein expression.

Figure 8

(A) Smad2 was more important than Smad3 in regulating VSMC marker gene promoter activity in NCCs. Monc-1 cells were transduced with adenovirus expressing GFP (shGFP), Smad2 (shS2), or Smad3 (shS3) shRNA for 2 days and transfected with SM22α promoter followed by vehicle (Ctrl) or TGF-β (5 ng/ml) treatment. Luciferase assays were performed. Luciferase activity was normalized to renilla activity. *P<0.001, **P<0.05 compared to shGFP/TGF-treated groups. (B) Smad2, but not Smad3, was essential for VSMC mark protein expression in NCCs. Monc-1 cells were treated similarly as described in A without promoter transfection. α-SMA and SM22α protein expression was detected by western blot. (C) Quantification of protein expression shown in B by normalized to α-tubulin. *P<0.01 compared to Ad-GFP group for α-SMA and SM22α, respectively. (D) Smad3 was more important than Smad2 in VSMC differentiation from mesenchymal progenitors. 10T1/2 cells were co-transfected with SM22α promoter and pcDNA, Smad2, or Smad3 cDNA followed by vehicle (Ctrl) or TGF-β treatment. Luciferase assays were performed. Luciferase activity was normalized to renilla activity. *P<0.01 compared to pcDNA/TGF-β-treated group. (E and F) Smad2 was deleted in SMCs by crossing Smad2floxed mice with SM22α-Cre mice. Carotid (CA) or thoracic artery (TA) media layers from wild type littermates (WT) or SMC-specific Smad2 knockout mice (KO) were homogenized, and total RNA was extracted. qPCR were performed to detect MMP2 (E) and PCNA expression (F) and fold inductions were shown. *P<0.01 compared to the corresponding WT SMCs. (G) MMP2 and PCNA protein expression in WT and Smad2 KO SMCs was examined by western blot. (H-I) MMP2 (H) and PCNA expression (I) was normalized to α-tubulin. *P<0.01 compared to the corresponding WT SMCs.