addgene pbabe bleo human pparγ2  (Addgene inc)

Journal: Molecular Metabolism

Article Title: A novel peroxisome proliferator-activated receptor gamma ligand improves insulin sensitivity and promotes browning of white adipose tissue in obese mice

doi: 10.1016/j.molmet.2021.101363

Figure Lengend Snippet: WO95E is a high affinity partial agonist of PPARγ. (A). Chemical structure of WO95E. (B). Binding affinity of WO95E to the ligand binding domain of PPARγ in in vitro competition binding assay. Fluorescence polarization value (mP) was measured at 485 nm (excitation) and at 535 nm (emission). The data shown are representative of 3 independent experiments. (C). Transactivation activity of compounds in HEK293 cells transfected with PPARγ2 and 3x PPRE-luc reporter. Transactivation activity was presented as fold change with that of Rosi designated as 1. The data shown are representative of 3 independent experiments. (D). Oil Red O staining in 3T3-L1 cells differentiated with differentiation media in the presence of compounds at 10 μM. (E). Relative mRNA expression levels of adipogenic genes in 3T3-L1 differentiated with differentiation media in the presence of compounds at 10 μM by qRT-PCT. The results are expressed as fold change and are representative of 3 independent experiments. Data are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: Plasmids pBabe bleo human PPARγ2 (#11439) and PPRE X3-TK-luc (#1015) were acquired from Addgene.

Techniques: Binding Assay, Ligand Binding Assay, In Vitro, Fluorescence, Activity Assay, Transfection, Staining, Expressing

Journal: Molecular Metabolism

Article Title: A novel peroxisome proliferator-activated receptor gamma ligand improves insulin sensitivity and promotes browning of white adipose tissue in obese mice

doi: 10.1016/j.molmet.2021.101363

Figure Lengend Snippet: WO95E inhibits PPARγ S273 phosphorylation. (A). Phosphorylation of PPARγ S273 in 3T3-L1 differentiated adipocytes treated with TNFα 10 ng/ml for 1 h in the presence of compounds at 10 μM pSer273 of PPARγ was detected by Western blotting using PPARγ pSer273-specific antibody. The data shown are representative of 3 independent experiments. (B). Quantification of Phosphorylation of PPARγ S273. Data are the mean ± SEM. ∗P < 0.05, and ∗∗∗P < 0.001 compared to DMSO control in the presence of TNFα. (C). Relative mRNA expression levels of pSer273-associated genes in 3T3-L1 differentiated with differentiation media in the presence of compounds at 10 μM by qRT-PCT. The results are expressed as fold change and are representative of 3 independent experiments. Data are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (D). Docking simulation of the PPARγ LBD:WO95E complex. WO95E is in red. (E). Schematic representation of atomic interaction between PPARγ LBD and WO95E. Hydrogen bonds are shown in brown dashed lines with donor-acceptance distances in angstroms. (F–G). Transactivation activity of compounds in HEK293 cells transfected with PPARγ2 and 3x PPRE-luc reporter, in the presence and absence of GW9662 (5 μM). Both Rosi (F) and WO95E (G) were tested at 10 μM. Transactivation activity was presented as fold change with that of DMSO as 1. The data shown are representative of 3 independent experiments. Data are the mean ± SEM. ∗P < 0.05. (H). Transactivation activities shown in (F–G) were plotted as ratio of a compound in the presence of GW9662 over that in the absence of GW9662. Data are expressed as mean ± SEM. ∗∗P < 0.01 compared to DMSO control group.

Article Snippet: Plasmids pBabe bleo human PPARγ2 (#11439) and PPRE X3-TK-luc (#1015) were acquired from Addgene.

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Activity Assay, Transfection