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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Int J Biochem Cell Biol. 2013 Apr 8;45(7):1274–1280. doi: 10.1016/j.biocel.2013.03.020

Figure 4.

Figure 4

Collagen inhibits canonical Notch signaling. (A) Effects of collagen on the SM22 promoter require its RBP-jK site, the target of canonical Notch signaling. A7R5 cells were transfected with a proximal SM22 promoter luciferase reporter construct prior to co-culturing with control or Notch ligand expressing cells. Cocultures were treated with increasing concentrations of type IV collagen. Smooth muscle promoter activation was determined using the ratio of firefly/Renilla luciferase activity. Collagen effectively inhibited expression of the wild type SM22 construct, but the mutant SM22-luciferase exhibited constitutive activation that was not affected by extracellular collagen. (B–F) A7R5 cells were transfected NICD from NOTCH3 to constitutively activate the Notch pathway in the absence of extracellular stimulation. Transfected cells were treated with type IV collagen, which did not repress NICD-stimulated reporter expression. Target gene regulation was assessed by quantitative RT-PCR of genes indicated in the Y-axis. RNA content was normalized to 18S control rRNA levels in each group. All groups included three replicates. * denotes significant differences between control and Jagged-stimulated cultures (p<0.05).