Fig. 5

The involvement of TonEBP/NFAT5 in glucose-induced endothelial COX-2 expression. a Western analysis of the effect of siRNA to TonEBP/NFAT5 on COX-2 expression in endothelial cells exposed to high glucose and high mannitol. Serum-starved human aortic endothelial cells (HAECs) were transfected with fluorescein isothiocyanate (FITC)-coniugated-siRNA to TonEBP/NFAT5 or siRNA to scrambled sequence for 24 h, and then treated with stimuli for 24 h with high glucose, or high mannitol, or equimolar concentration of sodium chloride. At the end of treatments transfection efficiency was checked by observing the plates under fluorescence microscopy. Protein expressions for cyclooxygenase (COX)-2 was detected by Western analysis, with β-actin serving as loading control. Data represent for each condition the mean ± S.D. from three separate experiments. **, P < 0.01 mannitol- or glucose-treated vs control HAECs; §, P < 0.05 vs without TonEBP/NFAT-siRNA. b The effect of hyperosmotic stress on NF-κB activation in human aortic endothelial cells. Electrophoretic mobility gel shift assay (EMSA), showing the effect of hypertonic stress on NF-κB activation. EMSA was performed by mixing the ³²P-oligonucleotide encoding for the NF-κB binding probe with nuclear extracts from osmotically stressed HAECs for 1–3 h. The electrophoretic run with nuclear protein extracts from unstimulated HAECs is shown in lane 1 and lane 7. The supershift analysis with a polyclonal rabbit antibody against p65, shows upward shift of binding complexes. Results are representative of three separate experiments. c Scanning densitometry of the shift bands (highlighted within the rectangle) of EMSA gels, expressed as arbitrary units of optical density. Data represent for each condition the mean ± S.D. from 3 separate experiments. *, P < 0.01 vs control HAECs