Figure 6. The transcriptional activities of CREB and NFAT5 contribute to induction of osmotic, but not hypoxic, expression of the OPN gene in RPE cells. The level of OPN mRNA was determined with real-time reverse transcription (RT)–PCR in cells cultured for 24 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and in the presence of CoCl₂ (150 µM), respectively. A: The following agents were tested: the STAT3 inhibitor Stattic (1 µM), the NF-κB inhibitor CAPE (5 µM), and a HIF-1 inhibitor (HIF Inh; 5 µM). B: Effects of the CREB inhibitor 666–15 (250 nM) and the AP-1 inhibitor SR11302 (5 µM) on the expression of the OPN gene. C, D: Effects of NFAT5 siRNA (siNFAT5; 5 nM) and nontargeted scrambled siRNA (siNon; 5 nM) on the levels of the NFAT5 mRNA (C) and OPN mRNA (D). The numbers of independent experiments using cell lines from different donors are indicated in the bars. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ^●p<0.05. Statistically significant difference versus CoCl₂ control: ^○p<0.05. Statistically significant difference versus nontargeted siRNA: ^♦p<0.05.