Fbs gold

Mouse keratinocyte cell lines were established following published protocols^{42 (link),43 (link)}. Briefly, keratinocytes were isolated from 8-wk-old female mouse dorsal skin and cultured on mitomycin C treated J2-3T3 feeders in type I collagen-coated flasks in DMEM-Ham’s F12 (3:1) medium (ThermoFisher) supplemented with 1.8 × 10−4 mol/l adenine, 0.35 mM calcium, 7.5% FBS Gold (Biowest), 100 U/ml penicillin/100 μg/ml streptomycin (Biowest), 2 mM glutamine (Biowest), 0.25 μg/ml amphotericin B (Biowest), 5 μg/ml insulin, 10⁻¹⁰M cholera toxin, and 10 ng/ml EGF (Peprotech). Following approximately 8 passages, spontaneously immortalized lines arose, which were maintained using the above conditions.
To evaluate response to GCs, cells were grown overnight in medium containing charcoal-stripped serum to deplete steroids, and then treated with vehicle (EtOH) or 100 nM Dex for indicated time periods. In specified experiments, cells were treated with 100 nM PMA, 10 μM BIRB196, or 5 μM U0126 (Merck Millipore).

Cultured keratinocyte cell lines were previously established from adult mouse epidermis^{34 (link)}. Briefly, keratinocytes were isolated from 8-wk-old female mouse dorsal skin and cultured on mitomycin C treated J2-3T3 feeders in type I collagen-coated flasks in DMEM-Ham’s F12 (3:1) medium (Thermo Fisher; Biowest, Nuaillé, France) supplemented with 1.8 × 10⁻⁴ mol/l adenine (Sigma), 0.35 mM calcium, 7.5% FBS Gold (Biowest), 100 U/ml penicillin/100 μg/ml streptomycin (Biowest), 2 mM glutamine (Biowest), 0.25 μg/ml amphotericin B (Biowest), 5 μg/ml insulin (Sigma), 10⁻¹⁰ M cholera toxin (Sigma) and 10 ng/ml EGF (Peprotech, Rocky Hill, NJ). Following approximately 8 passages, spontaneously immortalized lines arose.
Prior to treatments, cells were grown overnight in medium containing charcoal-stripped serum to deplete steroids, then treated with aldosterone, the GR antagonist RU486, or the MR antagonist eplerenone at indicated concentrations and times (all from Sigma). For combined treatments, cells were pre-incubated with either RU486 or eplerenone for 16 h and then treated with aldosterone for 6 h. When the two antagonists were used together with aldosterone, each reagent was used at 1 μM.