Selenium

Fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), propidium iodide, mitotane, and all other chemicals were purchased from Sigma Aldrich (Italy). DMEM-F12, 0.05% trypsin-EDTA, insulin, transferrin, selenium, and antibiotics were from Thermo Scientific (Milan, Italy). Adrenocortical tumor cells (H295R and SW13 cells) were obtained from the American Type Culture Collection (ATCC, Rockwille, MD). SW13 cells have a doubling time of about 24 h, H295R cells show a >48–72 h doubling time: this difference has been considered in cell experiments (Wang and Rainey, 2012 (link)). Furthermore, human fibroblasts derived from a donor were used as normal diploid human cells. Written informed consent to the collection and use of these cells for research purposes was obtained. All cells were cultured as previously described (Mariniello et al., 2012 (link)).

DRG neuron cultures were prepared from 5-week-old male C57BL/6 mice (Japan SLC) and 3-week-old Sprague Dawley rats (Japan SLC) as previously described [17 (link)]. In brief, DRGs were collected, dissociated by collagenase (Wako Pure Chemical) and diluted in a medium consisting of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 media (Thermo Fisher Scientific, Waltham, MA, United States of America), 17.5 mM glucose, and 30 nM selenium (Thermo Fisher Scientific). Isolated DRG neurons were seeded on glass coverslips coated with poly-L-lysine (Thermo Fisher Scientific). DRG neurons were cultured with or without 1 μmol/l ranirestat (Sumitomo Dainippon Pharma).
After 24 hours of culture, the cells were fixed with 4% (wt/vol) paraformaldehyde for 20 minutes and blocked with 1% bovine serum albumin (Wako Pure Chemical) for 30 minutes at 4°C. Thereafter, the DRG neurons were immunologically stained with rabbit polyclonal antineurofilament heavy-chain antibody (1 : 500; Merck) and visualized with Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (1 : 300; Life Technologies). Images were captured utilizing fluorescence microscopy Olympus IX73 (Olympus, Tokyo, Japan). Neurite outgrowth was observed in 10-20 neurons per coverslip and evaluated by ImageJ software (National Institutes of Health, Bethesda, MD, United States of America).

After thawing/warming, some ovaries were cultured for 4 hours at 37 °C in 96-well plates (see Table S1 for the experimental distribution). Culture medium was composed of Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Gibco, Waltham, MA, USA) supplemented with 10% decomplemented and desteroidized FBS, 200mM L-Glutamine (Thermo Fisher Scientific, Gibco, Waltham, MA, USA), 1% of a mixture of insulin (1000 mg/l), transferrin (550 mg/l) and selenium (0.67 mg/l) (Thermo Fisher Scientific, Gibco, Waltham, MA, USA), 1 μl/ml penicillin-streptomycin (Thermo Fisher Scientific Gibco 15140–122 Waltham, MA, USA), 100 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and 0.2% human FSH (Sigma-Aldrich, St. Louis, MO, USA).