To access the data, click or select the words “Appendix 3.” TLR9 gene transcription and mRNA stability. Hyperosmotic and CoCl₂-induced expression of the TLR9 gene is mediated by stimulation of gene transcription in cultured RPE cells. The relative mRNA levels were determined by qPCR and are indicated as fold of control (A, C) and as percent of the 0 h control (B, D), respectively. (A) Chemical hypoxia (150 µM CoCl₂, n=4) as well as (C) high extracellular osmolarity (+100 mM NaCl, n=4–5) induced TLR9 gene expression was inhibited by actinomycin D (ActD; 5 µg/ml) that blocks RNA polymerase II. RPE cells were pre-incubated with ActD for 30 min and then for further 6 h with and without NaCl or 24 h with and without CoCl₂ respectively. (B)TLR9 stability was not changed under CoCl₂-induced hypoxia or (D) high extracellular osmolarity compared to untreated controls in cultured RPE cells. The cells were pre-stimulated for 24 h with CoCl₂ (n=4), and for 12 h with NaCl (n=3–5) respectively. Then, ActD was added, and RNA was isolated at different time points. (B Inset) Ethanol as vehicle of ActD solution had no effect on TLR9 gene expression (n=4–5). Each bar shows data obtained in independent experiments with cells from different donors. ACTB and B2M were used to normalize the results obtained with CoCl₂ and ACTB was used to normalize the NaCl results. Mann–Whitney-U-Test was used for statistical analysis. Significant difference versus unstimulated control: *p<0.05 and to NaCl or CoCl₂-control, respectively ● p<0.05 (A, C). Significant difference versus 0 h control: ○ p<0.05 (B, D).