Collagenase type 2 c2 bioc

Male Sprague-Dawley rats (3 months old, weighing 250-300 g) from JOINN Laboratories were used for cell preparation. Rats were sacrificed by CO 2 asphyxiation and NP tissues were dissected immediately and rinsed with saline. Primary NPCs were isolated by enzymatic digestion using 10 mg/ml Collagenase Type II (C2-BIOC, Sigma-Aldrich) for 4 hours in a shaker at 37°C. NPCs were then centrifuged at 400 ×g for 5 minutes, suspended, and cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12, 11320033, Gibco, Grand Island, New York) medium containing 10% fetal bovine serum (16140071, Gibco) and 1% Penicillin-Streptomycin-Amphotericin B Solution (PB180121, Procell, Wuhan, China) at 37°C, in a humidified atmosphere containing 5% CO 2 . The siRNAs to deplete Yap or Ccnb1 were purchased from GenePharma (Shanghai, China) and details are listed in Supplementary Table . 3. According to the manufacturer's instructions, NPCs were seeded in 6-well plates at 70% confluency, and 100 nmol of each siRNA was transfected into cells in each well, using Lipofectamine 3000 (L3000001, Invitrogen, Waltham, Massachusetts). Otherwise, NPCs were treated with 0.25 μmol/L verteporfin or 5 μg/ml nocodazole (HY-13520, MCE, Shanghai, China) for 48 hours before collection.