Nci h295r cells

NCI-H295R cells (ATCC) at passages 5–8 were initially expanded and cultured in 2D plastic according to ATCC recommendations using DMEM:F12 medium containing 0.00625 mg/mL insulin, 0.00625 mg/mL transferrin, 6.25 ng/mL selenium, 1.25 mg/mL bovine serum albumin, 0.00535 mg/mL linoleic acid, and 2.5% Nu-Serum I (media supplements available from Corning). At 80% confluence, cells were harvested with 0.25% (W/V) Trypsin-0.53 mM EDTA solution (Thermo Fisher) and resuspended in media for 2D culture or 3D hydrogels.

Human adrenal NCI-H295R cells purchased from American Type Culture Collection (ATCC; CRL-2128) were maintained under normal growth conditions (growth medium, GM) in DMEM/Ham’s F-12 medium containing L-glutamine and 15mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (Becton Dickinson, Franklin Lakes, NJ USA), 0,1% insulin, transferrin, and selenium (100U/ml; GIBCO), 1% penicillin (100 U/ml; GIBCO) and streptomycin (100μg/ml; GIBCO). The serum-free NCI-H295R medium (starvation medium, SM) contained DMEM/Ham’s F-12 medium, penicillin (100 U/ml; GIBCO), and streptomycin (100 μg/ml; GIBCO) only. The cultures were kept at 37°C with 5% CO_2, and the cells were divided once a week. For steroid profiling, mRNA expression and protein expression experiments, cells with passage numbers 18 to 23 were subcultured for 24 hours in GM on 6-well plates and then starved, or alternatively treated with metformin and resveratrol. Metformin was dissolved in water and used at a final concentration of 1mM. Resveratrol was dissolved in DMSO and used at final concentrations of 5μM to 50μM.

All mammalian cells were grown under 37°C, 5% CO₂ incubation conditions. Female NCI-H295R cells were purchased from American Type Culture Collection (ATCC) in September 2019 (CVCL_0458) and were maintained in ATCC Dulbecco’s modified Eagle’s medium (DMEM):F12 medium containing 2.5% Corning NuSerum I and 1% Corning ITS+ supplement. Male ATC7L cells were maintained in Gibco DMEM:F12 with added 5 ml l-glutamine (200 mM) and 5 ml of Gibco ITS supplement (79 (link)). Virus was produced in female HEK-293T cells purchased from Dharmacon in 2015 and grown in DMEM (Gibco) medium with 10% fetal bovine serum (HyClone). HEK-293T cells for expression and purification of affinity-purified recombinant PKA proteins were cultured in DMEM (Lonza) supplemented with 10% fetal bovine serum (HyClone), penicillin (50 U/ml), and streptomycin (0.25 μg/ml; Lonza).

Human adrenocortical NCI-H295R cells were obtained from American Type Culture Collection (ATCC; CRL-2128). H295R cells were cultured in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (BD Biosciences, Allschwil, Switzerland), 0.1% insulin, transferrin, and selenium (100 U/ml; GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 µg/ml; GIBCO). The serum-free starvation medium consisted of DMEM/Ham’s F-12 medium, penicillin and streptomycin (100 µg/ml; GIBCO). For microarray experiments and for steroid profiling experiments, cells were first grown in normal growth medium for 24 h. Medium was then replaced, and cells were grown in the presence of 10 mM metformin in serum-free medium for 48 h^{21 (link)}.

Human adrenocortical NCI-H295R cells were purchased from American Type Culture Collection (ATCC; CRL-2128). H295R cells were cultured in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (BD biosciences), 0.1% insulin, transferrin, and selenium (100 U/ml; GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). The serum free starvation medium consisted of DMEM/Ham’s F-12 medium, penicillin and streptomycin (100 μg/ml; GIBCO). Mouse Leydig (MA-10) cells were kindly provided by Prof. Brigitte M. Frey, Bern, Switzerland. MA-10 cells were cultured in Waymouth medium (Sigma–Aldrich) and supplemented with 15% horse serum (GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). For MA-10 cells, culture dishes were pre-coated with 0.1% gelatin (Sigma–Aldrich). The serum free starvation medium of MA-10 consisted of Waymouth medium and antibiotics. Charcoal treatment of NU-I serum was performed by adding charcoal-dextran coated powder (1 g) to NU-I serum (50 mL) while gently mixing on a shaker table overnight. The followingday, charcoal-stripped serum was obtained by centrifugation at 2’000 g for 15 minutes and filtration through a 0.2 μm filter.