Nerve growth factor (ngf)

DRG neurons were isolated from male and female wild-type or Gα_q^−/− mice of 2-7 days after birth following sacrifice by cervical dislocation and cultured as described previously (Than et al., 2013 (link), Zhang et al., 2008 (link)). Briefly, isolated DRGs were treated with type IV collagenase (Worthington, LS004186) followed by trituration. The dissociated DRG neurons were then seeded onto coverslips coated with poly-L-lysine (Sigma, P9155) and laminin (BD, 354232) and maintained in DMEM media (Thermo fisher, 11564446) containing 10% fetal bovine serum (Thermo Fisher, 11550356), 2mM L-glutamine, 100IU/ml penicillin, 100 μg/ml streptomycin supplemented with 5 μM cytosine β-D-arabinofuranoside (Sigma, C1768) and 50ng/ml nerve growth factor (Promega, G5141). The cultured DRG neurons were used within 24 hours after isolation.
HEK293 cell line was purchased from ATCC, and MEF cells derived from Gα_q/11^−/− mice were obtained from Prof. Stefan Offermanns (Max-Planck-Institute, Germany) as described previously (Zhang et al., 2012 (link)). The cells lines were maintained in high glucose DMEM media containing 10% fetal bovine serum, 100IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator (5% CO2). Cells were used between 15 and 30 passage numbers. The cell lines were derived from embryos and the sex of these cell lines is thus not available.

DRG were collected and placed into F-12 Nutrient Mixture (Ham; Gibco) supplemented with 0.1% collagenase type IV (Worthington). Thereafter, DRG were triturated and cell suspensions centrifuged at 600 RPM for 6 min. Pellets were resuspended in fresh DRG medium supplemented with NGF (10 ng/ml; Promega), and plated on poly-L-ornithine (100 µg/ml, Sigma-Aldrich)-pre-coated glass coverslips. Cultures (10,000–22,500 cells/well) were incubated at 37 °C for 24 h. In the experiments where miR-21 was overexpressed, cultured neurons were transduced with a green fluorescent protein (control) or miR-21 lentiviral vector^{24 (link)} and were incubated at 37 °C for 72 h.