M7898

SCs were cultured and purified as previously described (Gu et al. 2014 (link)). Sciatic nerves were harvested from neonatal rats (1–2 days), cut into small pieces and enzymatically treated with 1% collagenase (17018-029, Gibco, Carlsbad, CA, USA) and 0.125% trypsin (25200-056, Gibco). The cell mixture was then resuspended in DMEM (11965-092, Gibco), 10% FBS (12483-020, Gibco), and penicillin–streptomycin (PS; 15140122, Thermo Fisher Scientific, Cleveland, OH, USA) using a 400-mesh cell screen to make a single-cell suspension, seeded in 50 μg/ml poly-d-lysine (PDL; P-7890, Sigma, St Louis, MO, USA) coated dishes with 10 mM cytosine arabinoside (C6645, Sigma) and 10% FBS in DMEM for 1 day to remove fibroblasts. Afterwards, the medium was changed to supplemented with 5 μM forskolin (F6886, Sigma), 2 ng/ml Neuregulin 1 (Nrg1; 396-HB-050, R&D, MN, USA) and 10% FBS in DMEM. After 48 h, cells were incubated with anti-Thy1 antibody (1:1000, M7898, Sigma) to remove remaining contaminating fibroblasts. SC purity was assessed by S100β (1:500, S2532, Sigma) immunostaining. Passage 2 SCs with > 95% purity were used for all cell experiments.

Primary Schwann cells were collected from sciatic nerves of neonatal SD rats, purified with anti-Thy1.1 (1:1000, M7898, Sigma) and rabbit complement (Invitrogen, Carlsbad, CA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; 10-013-CVR, Corning, NY, USA) containing 10% fetal bovine serum (FBS; 10099141c, Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (c0222, Beyotime, Shanghai, China), 2 μM forskolin (Sigma), and 10 ng/ml β-heregulin (HRG; R&D Systems Inc., Minneapolis, MN, USA) in a humidified 5% CO₂ incubator at 37 °C. For LIF or EREF knockdown, cultured primary Schwann cells were transfected with siRNA segments targeting LIF (siRNA-1 sequence: GCTCATTCTGCACTGGAAA and siRNA-2 sequence: ATGCCAATGGGACAGAGAA), a siRNA segment targeting ERFE (sequence: TGAAGGAGTTCCAGTTGTT), or a non-targeting negative control (random sequence, RiboBio, Guangzhou, Guangdong, China) for 48 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen). For LIF overexpression, cultured primary Schwann cells were transfected with LIF-overexpressing-lentivirus (pLenti-CMV-EGFP-P2A-LIF-3FLAG) or a negative control lentivirus (pLenti-CMV-EGFP-P2A-MCS-3FLAG) (OBiO, Shanghai, China) for 72 h.

SCs were isolated from the sciatic nerves obtained from dissected sciatic nerves of 2-day-old rats, minced, incubated in 3 mg/mL collagenase for 30 min at 37 °C, followed by trypsinization at 37 °C for 8 min. Primary cultures of SCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 g/mL streptomycin at 37 °C in a 5% CO₂ humidified atmosphere. Primary cultures were then treated with cytosine β-D-arabinofuranoside (C1768, Sigma-Aldrich) at 10 μM to remove fibroblasts. The viable fibroblasts were eliminated by complement cleavage of polyclonal anti-Thy1.1 antiserum (1:1000, M7898, Sigma-Aldrich) and rabbit complement (234,400, EMD Millipore). The final cells consisted of 98% SCs were determined by immunofluorescence for mouse anti-S100β (S2532, Sigma-Aldrich) monoclonal antibody which is a specific SC marker.