Fig. 5.
TEAD1 promotes embryonic VSMC differentiation. a. Using a flow cytometry, VSMCs were isolated from the dorsal aortae of E13.5 Tagln-Cre−/Tead1F/F/mTmG+/−, Tagln-Cre+/Tead1F/W/mTmG+/−, and Tagln-Cre+/Tead1F/F/mTmG+/− embryos and cultured to directly visualize GFP positive VSMCs. b Isolated VSMCs from the dorsal aorta of E13.5 control or Tead1 KO embryos were cultured and total RNA was extracted. qRT-PCR was performed to assess relative changes in gene expression as indicated. The level of gene expression in control VSMCs was set to 1 (red line). Data were obtained from cells cultured from 6 embryos per genotype. *p < 0.05. c Primary aortic VSMCs isolated from control or Tead1 mutant embryos were transduced with adenovirus encoding control GFP or TEAD1 and the expression of smooth muscle-specific genes was analyzed by Western blot. Restoration of TEAD1 expression in Tead1-null VSMCs rescues the differentiation defects seen in Tead1-deficient cells. d Densitometric analysis of protein expression in “C” normalized to the loading control VCL and relative to signals from control cells infected with GFP (set to 1, red line). N = 4. *p < 0.05, vs. Control + Ad-GFP group; #p < 0.05, vs. KO + Ad-GFP group. e Cultured primary VSMCs from E13.5 TEAD1F/F embryos were transduced with control GFP or Cre adenovirus and subsequently cells were treated with or without TGF-β, an established driver for VSMC differentiation. 48 h posttransduction, cells were harvested for analysis of smooth muscle-specific gene expression by Western blot. f Densitometric analysis of protein expression in “E” normalized to the loading control TUBA. *p < 0.05 and &p < 0.05, vs. cells infected with GFP and no TGF-β treatment (set to 1, red line); #p < 0.05, vs. cells infected with GFP and with TGF-β treatment. KO of endogenous TEAD1 expression impairs TGF-β induced smooth muscle-specific gene expression. g Luciferase reporters driven by the promoter regions of smooth muscle-specific genes, Tgfb1i1 or Lmod1 were transfected into control or Tead1 KO primary embryonic VSMCs and dual luciferase assays were carried out to determine the degree of promoter activity in transfected cells. Promoter activity in control cells was set to 1 (red line). N = 6. *P < 0.05. h Smooth muscle-specific luciferase reporters (Tgfb1i1 or Lmod1) were transfected into control embryonic VSMCs with or without co-transfection of a TEAD1 expression plasmid. At 36 h posttransfection, the effects of TEAD1 on promoter activity were determined using dual luciferase assays. Baseline promoter activity without TEAD1 co-transfection was set to 1 (red line). N = 3–6. *P < 0.05