5 oxo ete

H295R, an adrenal cell line with a steroid secretion pattern similar as that of freshly isolated adrenocortical cells [36 (link)–38 (link)], was purchased from ATCC and cultured in Dulbecco’s modified Eagle’s/F12 medium (DMEM/F12, Gibco, Life Technologies) supplemented with 5% bovine Cosmic calf serum (HyClone, GE), 1% ITS+1 (Sigma), 200 UI/ml penicillin, and 200 μg/ml streptomycin sulfate (Gibco, Life Technologies) at 37 °C and 5% CO₂. For ectopic expression of the OXE-R in H295R cells, a 1.5-kb fragment of OXER1 cDNA was cloned from this cell line and inserted into pBABE to generate pBABE-OXER1. This construct was used for the generation of the stable cell line H295R-OxeR1, as described elsewhere [35 (link)].
The following reagents were used: 5-oxo-ETE (Santa Cruz), 8Br-cAMP (Sigma), angiotensin II (Sigma), phorbol 12-myristate 13-acetate (PMA, LC Laboratories), zileuton (5-LOX inhibitor, Cayman Chemical), H89 (PKA inhibitor, Calbiochem), PD98059 (MEK1/2 inhibitor, Calbiochem), SB203580 (p38 inhibitor, Cell Signaling Technology), wortmannin (PI3K inhibitor, Sigma), Gö6976 (inhibitor of classical PKCs, Tocris Bioscience), GF109203X (“pan” PKC inhibitor, Tocris Bioscience). Concentrations are indicated in the corresponding figure legends. 5-oxo-ETE is provided by Santa Cruz at a concentration of 0.3 mM in ethanol (vehicle).

Eicosanoids and/or inhibitors (from Cayman Chemical) were applied at the following concentrations: 1.0 nM 5-HETE (#34210), 1.0 nM 5-oxo-ETE (#34250), 7.5 nM MK886 (#21753), 25 μM Gue1654 (#29686), 100 nM physcion (Santa Cruz Biotech SC-205805). Cells were allowed to equilibrate with inhibitors for 30 minutes before experimentation.