Fig. 4
The effect of high glucose and high mannitol on TonEBP/NFAT5 expression and activity in endothelial cells. a Western analysis of TonEBP/NFAT5 in endothelial cells exposed to high glucose and high mannitol. Serum-starved HAECs were transfected with fluorescein (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. At the end of treatments, transfection efficiency was checked by observing the plates under fluorescence microscopy. Protein expression for TonEBP/NFAT 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, c, d Recruitment of TonEBP/NFAT5 to TonE DNA binding sites in osmotically stressed human aortic endothelial cells. b Expression and distribution of TonEBP/NFAT5 in osmotically stressed HAECs for 1 h. Nuclear (N) and cytosolic (C) extracts were analyzed by Western blotting, using monoclonal anti-rabbit Ton antibody, with anti-lamin B serving as a loading control. c Electrophoretic mobility gel shift assay (EMSA), showing the effect of hypertonic stress on TonEBP/NFAT5 activation. EMSA was performed by mixing the 32P-oligonucleotide encoding for the TonEBP/NFAT5 binding probe with nuclear extracts from osmotically stressed HAECs for 1 h. Electrophoretic runs with nuclear protein extracts from unstimulated HAECs are shown in lanes 2 through 7. The run with a mutated DNA sequence is shown in lane 1. Results shown here are representative of three separate experiments. d Scanning densitometry of the EMSA gels, expressed as arbitrary units of optical density. Data represent for each condition the mean ± S.D. from three separate experiments. *, P < 0.01 mannitol- or glucose-treated vs control HAECs