Figure 1. Regulation of cyclooxygenases -1 and -2 (COX1 and COX2) gene expression in retinal pigment epithelial (RPE) cells. A: Presence of COX1 and COX2 transcripts in RPE cells. To confirm the correct lengths of the PCR products, agarose gel electrophoresis was performed using products obtained from two RPE cell lines (1, 2) derived from different post-mortem donors. Negative controls (0) were done by adding double-distilled water instead of cDNA as the template. The β-actin (ACTB) mRNA level was used to normalize the COX1 and COX2 mRNA levels. B–H: COX1 and COX2 mRNA levels, as determined with real-time reverse transcription (RT)–PCR after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars). The mRNA levels are expressed as folds of unstimulated control. B: Effects of extracellular hyperosmolarity induced by addition of high (+ 100 mM) NaCl on the COX1 and COX2 gene expression levels. C, D: Dose-dependencies of the effects of high extracellular NaCl on the COX1 and COX2 mRNA levels. Ten to 100 mM NaCl were added to the culture medium, as indicated in the bars. The data were obtained after stimulation of the cells for 6 h (C) and 24 h (D). E: Effect of extracellular hypo-osmolarity (Hypo; 60% osmolarity) on the COX2 gene expression level. F: Effect of the addition of 200 mM sucrose on the COX2 gene expression level. G: Effects of hypoxia induced by cell culture in 1% O₂, and by addition of 150 µM CoCl₂, on the COX2 gene expression level. H: Effects of inflammatory and growth factors on the expression of COX2. The following factors were tested: endothelial growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), placental growth factor-2 (PlGF-2), pigment epithelium-derived factor (PEDF), interleukin-1β (IL-1β), and matrix metalloproteinase-2 (MMP-2). Each factor was applied at 10 ng/ml. Each bar represents data obtained in three to ten independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to six donors; in total, cell lines from 21 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05.