To verify the role of PPARγ activation in the inhibition of lox-1 expression, semi-confluent HSCs were co-transfected with pLOX-1-Luc and the plasmid pPPARγ, or the plasmid pdn-PPARγ, at indicated doses. The cDNA expression plasmid pPPARγ contained a full size of wild-type PPARγ cDNA 24. pdn-PPARγ contained a full length of cDNA encoding dominant negative PPARγ (dn-PPARγ) 32. A total of 4.5 μg of plasmid DNA per well was used for co-transfection of HSCs in 6-well culture plates. It included 2 μg of pLOX-1-Luc, 0.5 μg of pSV-β-gal, and 2.0 μg of pPPARγ, or pdn-PPARγ, at indicated doses plus the empty vector pcDNA. The latter was used to ensure an equal amount of total DNA in transfection assays. After recovery, cells were treated with or without curcumin (20 μM) in DMEM with FBS (10%) for 24 hr. Prior experiments have suggested that 10% of FBS in the medium contains enough agonists to activate PPARγ in HSCs 19, 23, 24. Luciferase activity assays demonstrated that forced expression of wild-type PPARγ cDNA dose-dependently reduced luciferase activities (Fig. 5A). In contrast, forced expression of dn-PPARγ dose-dependently abrogated the inhibitory effect of curcumin on the lox-1 promoter activity (Fig. 5B). These results collectively suggested the inhibitory role of PPARγ activation and its requirement in the curcumin-caused inhibition of the lox-1 promoter activity.