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. 2023 Apr 8;12(8):1116. doi: 10.3390/cells12081116

Figure 3.

Figure 3

Reporter assays for AU9 PPARδ and PPARγ activity. Stable HEK293T cell lines expressing (A) PPAR-δ ligand binding domain driven GAL4 reporter assays determined AU9 activity when compared to increasing concentrations of full PPARδ agonist GW0742. (B) AU9 induces partial human PPAR-δ activity when compared to full PPARδ agonist GW074 by activating the PPARδ Response Element (DRE) via transient co-transfection into HEK293 cells with human PPARδ expression vectors along with the reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with AU9 (10 μM) and Pio (10 μM) for 24 h. Luciferase activity was normalized to Renilla luciferase activity as described in the Methods section. Values were based upon normalized luciferase activity and ΔΔct values shown as a fold change from control. Statistical values were obtained using two-tailed, unpaired t-test analysis ± S.E.M. Where n = 6 independent experiments with three replicates per experiment, *** p < 0.0001. (C) Bubble plot of qPCR analysis (fold change from control) of wild type and 3xTgAD mice treated with and without AU9 for three months daily (5 mg/Kg). (D) Stable HEK293T cell lines expressing PPARγ ligand binding domain driven GAL4 reporter assays determined AU9 activity compared to increasing concentrations of full PPARγ agonist Rosi. (E) AU9 induces human PPARγ activity by activating the AP2 response element via co-transfection into HEK293 cells transiently with human PPARγ vector with the reporter plasmid (AP2-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. (F) AU9 induces human PPARγ activity by activating the 3XPPRE-pk-Luc response element via co-transfection into HEK293 cells transiently control reporter plasmid (pk-Luc) with Renilla vector for 24 h. (G) Human PPARγ with tyrosine-473 substituted with phenylalanine demonstrates activity using the 3XPPRE-pk-Luc response element via co-transfection into HEK293 cells transiently control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with AU9 (10 μM), and Pio (10 μM) for 24 h. Luciferase activity was normalized to Renilla luciferase activity as described in the Methods section. Values for G and H were based upon normalized luciferase activity and fold change from control. Statistical values were obtained using two-tailed, unpaired t-test analysis ± S.E.M. Where n = 6 independent experiments with three replicates per experiment. *: p < 0.05 and ***: p < 0.0001.