Cortisol

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.

Cells were washed once in phosphate-buffered saline (PBS), and medium was changed 75 min before harvest. Harvested medium samples were snap-frozen until assayed. To assay, samples were diluted and subjected to measurement using Cayman Cortisol or Corticosterone ELISA kits. Values were determined using an absorbance plate reader and subsequent curve fit analysis. All experiments were completed in at least three separate biological replicates. Measurements were normalized to control level(s) within each replicate.