Figure 1. Regulation of AQP8 gene expression in cultured human RPE cells. A: Presence of AQP8 gene transcripts in RPE cells, as determined with RT–PCR analysis. The correct lengths of the RT–PCR products were confirmed with agarose gel electrophoresis using products obtained from cell lines of four different donors (1‒4). Negative controls (0) were performed with double-distilled water instead of cDNA as the template. ACTB mRNA was used as loading control. B: Effects of media which were made hyperosmotic with the addition of 100 mM NaCl or 200 mM sucrose, or with the coaddition of both agents, on AQP8 gene expression. The level of AQP8 mRNA was determined with real-time RT–PCR analysis in cells cultured for 2, 6, and 24 h (as indicated by the panels of the bars), and is expressed as folds of unstimulated control. C: Dose-dependency of the effect of high extracellular NaCl on the AQP8 mRNA level. Ten to 100 mM NaCl were added to the culture medium, as indicated in the bars. D: Effect of extracellular hypo-osmolarity (Hypo; 60% osmolarity) on the cellular level of AQP8 mRNA. E: Effects of the hypoxia mimetic CoCl₂ (150 µM), cell culture in a 0.2% O₂ atmosphere, and oxidative stress induced by addition of H₂O₂ (20 µM) on expression of the AQP8 gene. F: Effects of high (25 mM) glucose, triamcinolone acetonide (Triam; 50 µM), and exogenous VEGF (10 ng/ml) on AQP8 gene expression. Each bar represents mean ± standard error of the mean (SEM) obtained in four to ten independent experiments using cell lines from different donors. Significant difference vs. unstimulated control: *p<0.05. Significant difference vs. NaCl and sucrose: ^●p<0.05.