Figure 4. Transcription factor activity involved in mediating NaCl-induced expression of the AQP8 gene in RPE cells. The mRNA levels were determined with real-time RT–PCR analysis in cells cultured for 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media (as indicated by the panels of the bars), and are expressed as folds of unstimulated control. A: The following agents were tested: an HIF-1 inhibitor (HIF-Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), the NF-κB inhibitor CAPE (5 µM), the AP-1 inhibitor SR11302 (5 µM), and the CREB inhibitor 666–15 (250 nM). B, C: Effects of knockdown of NFAT5 gene expression with siRNA (siNFAT5; 5 nM) on NFAT5 (B) and AQP8 mRNA levels (C) in cells cultured for 6 h in iso- or hyperosmotic media compared to non-transfected cells (control). As negative control, nontargeted siRNA (siNon; 5 nM) was used. siRNA transfection was performed 50 h before the addition of 100 mM NaCl. Each bar represents mean ± standard error of the mean (SEM) obtained in three to 16 independent experiments using cell lines from different donors. Significant difference vs. unstimulated control: *p<0.05. Significant difference vs. NaCl control: ^●p<0.05. Significant difference vs. nontargeted siRNA: ^♦p<0.05.