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. 2018 Apr 13;40(12):967–978. doi: 10.1093/eurheartj/ehy188

Figure 4.

Figure 4

Deletion of delta-like homologue 1 (Dlk1) promotes TGF-β1/Smad3 activation and induction of extra-domain A-fibronectin in mice. (A) (upper panel) Phosphorylated Smad-3 immunostaining (green) in wild-type and delta-like homologue 1-null myocardium. WGA (magenta) stains membrane/extracellular matrix. White arrows indicate cardiomyocyte Smad-3 activation. Yellow arrows point to non-myocyte (i.e. fibroblast) Smad-3 activation. Representative western blots and ratio quantification of phosphorylated Smad-3/total Smad-3 in heart lysates (lower panel). (B) Extra-domain A-fibronectin immunostaining (red) in wild-type and delta-like homologue 1-null cardiac tissues. Double immunostaining for extra-domain A (red) and α-smooth muscle actin (green) identified a group of α-smooth muscle actin-positive cells (i.e. myofibroblasts) embedded in an extra-domain A-rich extracellular matrix (lower panel, right image). (C) Western blots and ratio quantification of extra-domain A fibronectin, α-smooth muscle actin, mTOR, and SF2 from wild-type and delta-like homologue 1-null hearts. Calsequestrin, actin, and Ponceau Red are loading controls. (D) qRT-PCR showing up-regulation of α-smooth muscle actin mRNA in delta-like homologue 1-null hearts. Scale bars: (A) 20 µm; (B) 40 µm (left panel), 20 µm (right panel). qRT-PCR, quantitative real time polymerase chain reaction; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; TGF, Transforming growth factor; WGA, Wheat germ agglutinin; MMP9, Matrix metallopeptidase 9.