Figure 6. PACA as a covalent activator for PPAR-γ. (A) Docking of OXO-PACA to PPAR-γ LBD. OXO-PACA was docked to PPAR-γ LBD (PDB:) by Autodock vina. (B) Amino acid residues for recognizing OXO-PACA. Hydrogen bonds are shown in green, whereas pi-alkyl interactions are shown in pink. (C) Luciferase assay for PPAR-γ activation. RAW264.7 macrophages were transiently transfected with PPRE-X3-TK luciferase and pRL control using Effectene transfection reagent from Qiagen. Transfected macrophages were treated with PACA for 24 h. Luciferase activities were measured using the Dual-Luciferase reporter assay system. Data were expressed as mean ± SD (n = 5). * p<0.05, **, p<0.01 (PACA+LPS vs LPS alone). (D–F) PPAR-γ dependence in PACA-regulated expression of macrophages biomarkers. RAW264.7 cells were pretreated with 10 μM GW9662 for 1 h, treated with 50 μM PACA for 2 h, and stimulated with 1 μg/mL LPS for another 24 h. iNOS and Arg1 were determined by Western blotting analysis. The blots were quantified by a densitometric method. The mRNAs for M2a biomarkers (Ym-1 and FZZ1) and M1 biomarker (IL-1β) were determined by qRT-PCR. n = 3, * p<0.05, **, p<0.01, ***, p<0.01 (PACA+LPS vs LPS alone). &, p<0.05, &&, p<0.01, &&&, p<0.001 (PACA+LPS vs LPS +PACA+GW9662).