Jian Wang et al. npg 523 Figure 6 PPAR-γ is a target of miR-27b in cardiomyocytes. (A) Dual luciferase activity assay of HEK293 cells cotransfected with a luciferase reporter plasmid containing the naive or mutant PPAR-γ 3′-UTR and a miR-27b or miR-24 expression plas- mid or a miR-27b inhibitor. *P < 0.05 relative to respective controls. + Represents 200 ng/well and ++ represents 400 ng/well. (B) RT-PCR and real-time PCR showed that miR-27b did not affect the mRNA level of PPAR-γ. (C) Western blots for PPAR-γ proteins from HEK293 cells and cardiomyocytes infected with Ad-miR-27b or Ad-anti-miR-27b. Right: quantitative analysis of fold change in expression of PPAR-γ. (D) Western blot showed that PPAR-γ level was decreased in the heart tissues of Smad4-deficient mice. Right: quantitative analysis of fold change in the expression of Smad4 and PPAR-γ. (E) PPAR-γ was decreased in the heart tissues of miR-27b transgenic mice revealed by western blot. GAPDH was used as a loading control. Lower: quantitative analysis of fold change in the expression of PPAR-γ. (F) PPAR-γ overexpression rescued hypertrophic growth of cardiomyocytes caused by miR-27b overexpression. Upper: western blot for PPAR-γ proteins from cardiomyo- cytes infected with Ad-PPAR-γ or Ad-GFP. Lower: Quantitative analysis of fold change in mean cell surface area of α-actinin- immunostained cardiomyocytes infected with adenoviruses (n = 160 cells/condition). *P < 0.05 relative to Ad-GFP. aP < 0.05 vs miR-27b overexpression. www.cell-research.com | Cell Research