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Figure 4 Pioglitazone Induces Known PPAR Target Gene Expression and PPAR-LBD Activation in ECs

(A) Northern blot analysis in HSVECs was performed for the PPAR target gene acyl-CoA-oxidase (ACO) and compared with GAPDH in HSVEC pre-treated (16 h) with pioglitazone or WY14643 at the concentrations shown before TNF stimulation. (B) Western blot analysis for IB expression was performed on total protein extracts (50 µg) from HSVECs treated with either pioglitazone (10 µM) or WY14643 (250 µM) before stimulation with human TNF. (C) Standard LBD activation assays were performed in bovine aortic endothelial cells stimulated with pioglitazone at the concentrations shown (0.01 to 100 µM). (D) PPAR-ligand binding domain (LBD) assays were done as before but responses were compared in NIH/3T3 (fibroblasts), HEK293 (human kidney epithelial), Hep-G2 (hepatic), and bovine aortic endothelial cell lines before stimulation with pioglitazone or WY14643 (both 10 µM). Values are expressed as luciferase/β-Gal activity mean ± SD (n = 3, *p < 0.05 bovine aortic endothelial cells vs. NIH/3T3, *vs. HEK293, ***vs. Hep-G2, both Student t and Mann-Whitney U tests). Abbreviations as in Figure 1.