Redox Regulation of PPARγ in Polarized Macrophages

Figure 4

Site-directed amino acid exchange of hPPARγ cysteines inhibits PPARγ-dependent transactivation. A PPARγ-dependent transactivator assay was used to verify the effect of LC/MS detected (C126, C129, C160, and C163) and potential (C284) redox-modified hPPARγ cysteines. Therefore, HEK293T cells were transiently transfected with vectors encoding hPPARγ wt and cysteine-to-alanine mutants and a Firefly luciferase under the control of a PPARγ-responsive element as promoter. Affiliation of the cysteines towards zinc finger domain (ZF) and ligand-binding pocket (LBP) are depicted. Cells were cotransfected with a Renilla luciferase vector with a permanent active promoter cloned in front of the gene, so that relative luminescence units (RLU) were calculated in a dual luciferase assay. Transfected cells were stimulated for 24 h with DMSO (a), rosiglitazone (1 μM), and GW9662 (10 μM) alone or in combination (b, c). are provided. DMSO treated hPPARγ wt cells were set as 1. (, , ).

(a)

(b)

(c)