Fig. 3. Induction of Ostf mRNA expression levels in primary gill cell culture. Primary gill cells were grown overnight in isotonic medium (320 mOsmol l^–1) and were then subjected to hypertonic treatments and/or actinomycin D (Act D) treatment. After 6 h (0.25 days) of treatment, total RNA was extracted and reverse transcribed for real-time PCR assay. (A) Hypertonicity was achieved by the addition of either (1) membrane-nonpermeable solute (NaCl) or (2) membrane-permeable solute (urea) to the medium, making its osmolarity 500 mOsmol l^–1. A significant induction of Ostf mRNA expression was observed in the hypertonic condition prepared by the addition of NaCl. (B) Effect of Act D on hypertonicity-induced Ostf mRNA expression. Hypertonicity and Act D cotreatment abolished the induced Ostf transcript levels. ^*P<0.05 compared with the control cells. The results were obtained from five independent experiments.