Figure 3.

The 5′ untranslated region (5′UTR) but not promoter of the neuropilin 2 (NRP2) gene is responsible for Smad3‐enhanced luciferase reporter activity.

A, Schematic of constructs containing the NRP2 gene promoter and/or 5′UTR. The β‐actin (ACTB) promoter is contained in the original Addgene luciferase reporter vector where no Smad‐binding elements are found, thus serving as a negative promoter control. B, Luciferase assay using aortic smooth muscle cells transfected with constructs listed in (A). Cells transfected with empty vector (EV) or Smad3‐overexpression (Smad3‐OE) were seeded in 96‐well plates and cultured for 12 hours. The cells were then transfected with the indicated luciferase constructs for 36 hours in basal medium and treated with transforming growth factor β (TGFβ1) (10 ng/mL) for 2 hours before adding the luciferase assay solution. Luciferase‐catalyzed bioluminescence reading was normalized to cell numbers in duplicate plates. Data are presented as mean±SD. Statistics: 2‐tailed Student t test, n=8; ***P<0.001; NS. The construct of NRP2 promoter alone without 5′UTR (PRO) served as a negative control for the 5′UTR activity. The construct of 5′UTR driven by the generic ACTB promoter where Smad3 does not bind served as a positive control. The similar results from this positive control and the construct of 5′UTR driven by the specific NRP2 promoter indicate a major contribution of the 5′UTR (vs the NRP2 promoter) to the Smad3‐mediated transcriptional activity, as measured through the luciferase reporter.