Fig. 2

The effect of siRNA to AQP1 on COX-2 expression in endothelial cells exposed to high glucose and high mannitol. Serum-starved HAECs were transfected with Cy5-coniugated-siRNA to AQP1 or non-coniugated-siRNA to a scrambled sequence for 24 h and then treated with stimuli for 24 h. At the end of treatments, transfection efficiency was checked by observing the plates under fluorescence microscopy (a), while AQP1 gene knockdown efficiency was detected at the protein level by Western analysis (b). Protein expression for COX-2 was detected by Western analysis, with β-actin serving as a loading control (c). LPS (1 μg/mL) and tumor necrosis factor (TNF)-α (10 ng/mL) were here used as positive controls for the expression of COX-2. Representative blots (c) are shown. The results of scanning densitometry (n = 3 independent experiments) are expressed as arbitrary units in panel d, where columns and bars represent the mean ± SD (**, P < 0.01 mannitol- or glucose-treated vs control HAECs; §, P < 0.05 vs without AQP1-siRNA)