Heregulin β1 hrg

Primary Schwann cells were isolated from the sciatic nerve segments of neonatal Sprague-Dawley (SD) rats as previously described [22 (link)]. Briefly, rat sciatic nerve segments were surgically excised and treated with collagenase and trypsin. Collected cells were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin and streptomycin (Invitrogen), 2 μM forskolin (Sigma), and 10 ng/ml heregulin β1 (HRG; Sigma) till confluence. Cultured cells were then treated with anti-Thy1.1 antibody (Sigma, St. Louis, MO, USA) and rabbit complement (Invitrogen, Carlsbad, CA, USA) to remove fibroblasts. Purified Schwann cells were grown in cell culture medium containing DMEM, 10% FBS, and 1% penicillin and streptomycin (Invitrogen) in a humidified 5% CO₂ incubator at 37 °C. Cultured primary Schwann cells were passaged for no more than 2 passages prior to use.

Human ovarian cancer cell lines, PEO1, SKOV3, and OVCAR3, were maintained in RPMI 1640 media (Gibco Invitrogen) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 μg/mL streptomycin, and 100 U/mL penicillin in an atmosphere of 5% CO₂ and incubated at 37°C. Before experimental treatments, cells were grown for 24 h in RPMI 1640 media prepared, but replacing FBS, with 5% double charcoal-stripped FBS (Fisher). Heregulin-β1 (HRG, Sigma) was used by preparing 1 μmol/L stock solution made with 5% trehalose and 10% FBS in phosphate-buffered saline (PBS) and diluted to a final concentration of 1 nmol/L with media during treatments. Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μM. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media. For ROS detection, 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma) solution was prepared with dimethylsulfoxide in amber tubes to a concentration of 50 mM and stored at −20°C in the dark until used. For the cytotoxicity assay, the CellTiter-Glo® 2.0 assay kit (Promega) was used: stored at −20°C or 4°C in the dark until use.