Figure 3.

TCF7L2 expression is directly associated with vascular smooth muscle cell apoptosis in vitro

HASMCs were infected with adenovirus (30 MOI)-encoding LacZ (control) or myc-tagged TCF7L2, and after 48 h, (A) the abundance of the indicated proteins in the cell extracts was determined by immunoblot (representative of three independent experiments) and (B) the mRNA expression of the indicated genes, relative to ACTB, by quantitative real-time PCR. A LacZ control was set at 1 as reference (n = 3; representative of three independent experiments). Data are mean ± SEM. ^∗∗p ≤ 0.0004. HASMCs were transfected with siRNA control or siRNA against TCF7L2 via Lipofectamine RNAiMax, and after 72 h, (C) the abundance of the indicated proteins and (D) the mRNA expression of the indicated genes were determined as described above with an siRNA control set at 1 as reference. ^∗∗p ≤ 0.0002. HASMCs were infected with adenovirus as in (A). After 48 h in serum-free medium, they were treated with Fas ligand (FasL, 100 ng/mL) in serum-free medium and apoptosis was assessed by (E) annexin V via FACS analysis after treatment for 4 h (n = 3; representative of three independent experiments) or (F) the abundance of the indicated proteins in the cell extracts after 6 h as determined by immunoblot (representative of three independent experiments). HASMCs were transfected with the indicated siRNA as in (C). After 72 h in serum-free medium, they were treated with FasL (100 ng/mL) for (G) annexin V assay after 4 h as in (E) or (H) immunoblot after 6 h as in (F) and subjected to immunoblot as in (E). Densitometry for (A), (B), (E), (F), and mRNA expression of BAX are provided in Figure S5.