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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 5. Smad2 was essential for MRTFB-mediated VSMC marker expression in NCCs.

Figure 5

(A-B) Smad2 was required for MRTFB-mediated VSMC marker gene transcription. Monc-1 cells were transduced with adenovirus expressing GFP (Ad-GFP), Smad2 (Ad-shS2), or Smad3 shRNA (Ad-shS3) and co-transfected with α-SMA (A) or SM22α (B) promoter construct and pcDNA or MRTFB cDNA as indicated followed by vehicle (Ctrl) or TGF-β treatment (5 ng/ml) for 16 hours. Luciferase assays were performed. Luciferase activity was normalized to renilla activity. *P<0.01, #P=0.043, &P=0.048 compared to Ad-GFP groups with TGF-β for both A and B, respectively. (C-D) Smad2 was required for MRTFB-mediated VSMC marker mRNA expression. Monc-1 cells were treated as in A and B but without promoter transfection. α-SMA (C) and SM22α (D) mRNA expression was detected by qPCR and normalized to GAPDH expression. *P<0.05, @P>0.05 compared to Ad-GFP group treated with TGF-β for both C and D. (E) Smad2 was required for MRTFB-mediated VSMC marker protein expression. Monc-1 cells were transduced with Ad-GFP or Ad-shS2 as indicated and treated with TGF-β. α-SMA and SM22α protein expression was detected by western blot. (F) Quantitative analysis of the protein expression in E by normalized to α-tubulin. *P<0.05 compared to Ad-GFP group for both α-SMA and SM22α, respectively.