Cortisol

Ten days after SW introduction, all fish from 6 of the 8 tanks were randomly netted, anesthetized in metomidate (∼6 mg L⁻¹) and intraperitoneally (i.p.) implanted with a slow release implant with or without cortisol (+F/-F) and returned to their respective tank (n = 36, density 22 kg m⁻³). The remaining two tanks were maintained as non-treated controls throughout the experiment.
Fish in the +F group received cortisol (Sigma-Aldrich Sweden AB, Stockholm, Sweden), at a dose of 50 µg g⁻¹ body weight (BW) dissolved in vegetable oil:vegetable shortening 1∶1, at a volume of 5 µL g⁻¹ BW and the -F group received the implant without cortisol at the same volume. The cortisol implants have been shown to result in increased circulating levels of cortisol in the fish up to one month [37] .
In the present study the cortisol implants resulted in a significantly increased plasma level of cortisol in the +F group compared to –F and non-challenged controls as reported previously [31] . In the +F group the average level of cortisol was 319.2 ± 46.9 ng mL^-1, in the –F group 9.7±2.9 ng mL^-1 and in the non-implanted control 6.7±1.9 ng mL^-1, respectively.

Amnion epithelial cells and fibroblasts were treated in phenol red‐ and FBS‐free culture medium. For RNA sequencing, fibroblasts were treated with or without cortisol (1 μM; Sigma; 24 h). To determine whether cortisol affected the expression of CEBPD in amnion epithelial cells, the epithelial cells were treated with cortisol (0.01–1 μM; 24 h). To examine whether cortisol, PGE2, and IL‐1β affected the expression of CEBPD, CEBPA, CEBPB, CEBPG, DDIT3, PTGS2, and HSD11B1 amnion fibroblasts, the cells were treated with cortisol (0.01–1 μM), PGE2 (0.01–1 μM; Cayman Chemicals, Ann Arbor, MI, USA), and IL‐1β (0.01–1 ng/ml; Sigma) for 24 h. To study the role of C/EBPδ in the regulation of COX‐2 and 11β‐HSD1 expression by cortisol, PGE2, and IL‐1β in amnion fibroblasts, the cells were treated with cortisol (1 μM) or PGE2 (1 μM) or IL‐1β (1 ng/ml) in the presence or absence of knockdown of C/EBPδ by siRNA for 24 h.

All of the DNA samples were ordered from Integrated DNA Technologies (Coralville, IA). The DNA sequences and modifications used are in Table 1. Surfactant, mercury acetate, SYBR Green I (SGI), thioflavin T (ThT), adenosine (ADE), and cortisol were purchased from Sigma-Aldrich. Half-area black 96-well microplates and human AB serum were purchased from Corning and Greiner. Quartz fluorescence cuvettes were purchased from Agilent. Several buffers were prepared. Buffer 1 (for most adsorption studies and Ade aptamer): 50 mM pH 7.6 Tris, 500 mM NaCl, 20 mM MgCl₂. Buffer 2 (for T₃₀ Hg²⁺ binding DNA): 20 mM pH 7.5 MOPS. Buffer 3 (for CSS.1-42 aptamer): 20 mM pH 7.5 HEPES, 100 mM NaCl, 10 mM MgCl₂. Buffer 4 (for Ade and Ade-M2 aptamer): 10 mM HEPES buffer, pH 7.6, 50 mM NaCl, and 4 mM MgCl₂. Buffer 5 (for Ade aptamer): 10 mM HEPES buffer, pH 7.6, 50 mM NaCl, 4 mM MgCl₂, and 5% human AB serum.