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. Author manuscript; available in PMC: 2010 Feb 27.
Published in final edited form as: Circ Res. 2009 Jan 15;104(4):466–475. doi: 10.1161/CIRCRESAHA.108.184846

Figure 3. Notch signaling is activated in mural cells upon coculture with endothelial cells.

Figure 3

(A) Luciferase reporter assay to assess Notch activity. HDFNs were transfected with pGL3-promoter-luciferase (pGL3) plasmid as control, or plasmid with 5 CBF1 binding elements upstream of the promoter (5XCBF1), cultured in the presence or absence of HUVECs, and luciferase activity was measured. RLU, relative light units. (B, C) qPCR analysis of Notch target genes, HEYL/HRT3 and HES1 in mural cells cultured with or without HUVECs. (D) Knockdown of NOTCH3 by siRNA in HDFNs, HUASMCs and HCASMCs was confirmed by qPCR and (E) Western blot analysis. (F) qPCR analysis of HEYL/HRT3 mRNA expression after NOTCH3 knockdown. Mural cells were transiently transfected with control siRNA (con) or NOTCH3 siRNA (N3). HUVECs were added, and cocultured for an additional 48 hours. * P < 0.05 relative to relevant control; ns, not significant.