Fig. 6.

Smad2 positively regulates AoSMC expression of FAS.

Human primary aortic SMCs (AoSMCs) were transfected with scrambled siRNA (Scr), Smad2-specific siRNA, empty vector (VEC), or Smad2 overexpression plasmid (Smad2-OE) for 12 h in basal medium (no FBS). The cells were cultured for another 12 h in fresh basal medium (no Lipofectamine) to recover, and then treated with solvent or 10 ng/ml TGFβ1 or 50 ng/ml PDGF-BB for 20 h before harvest for Western blot and qRT-PCR analyses.

A and B. Smad2 loss-of-function reduces FAS expression at protein and mRNA levels (Western and qRT-PCR analyses). C. Smad2 gain-of-function enhances FAS promoter activity (luciferase assay). D. Smad2 enrichment at FAS promoter regions (ChIP-qPCR).

Quantification: Densitometry of Western blots (similar ECL exposure) from independent repeat experiments was normalized (to GAPDH) and then averaged to calculate mean ± SEM, n = 3 independent experiments. Readings of triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged to calculate mean ± SD (n = 3 repeats). a.u., arbitrary unit.

Statistics: One-way ANOVA/Bonferroni post-hoc test; Student’s t-test was performed in C; **P < 0.01, ***P < 0.001.

E. Schematic working model of Smad2 regulations of MET and FAS that promote SMC apoptosis. While Smad2 partnering with P53 represses the transcription of MET, it also activates the transcription of FAS. The sum of decreased Met (anti-apoptotic) and increased Fas (pro-apoptotic) prompts SMC apoptosis.