Dulbecco s modified eagle s medium dmem f12

The hNPCs were passaged when 80% of the dish was covered. The first three generations of these cells were used for the experiments. For the organ culture of mouse intervertebral discs, mice under carbon dioxide anesthesia were sacrificed by cervical dislocation, and soaked in 75% alcohol for 5 min before sterile operation on a clean bench. Segments were removed from the lumbar spines of the mice with back-cuts. The intervertebral discs L2-3 and L3-4 (including the upper and lower endplates) were dissected and cultured on a 6-well plate, with 4 ml culture medium added to each plate. The hNPCs and mouse intervertebral discs were cultured in an incubator containing 5% CO₂ and at 37̊C, with the medium changed every other day. The culture media for the hNPCs is the special media purchased from ScienCell Research Laboratories (cat. no. 4801). The culture media for the mouse intervertebral discs contained 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 1% double-antibiotic (streptomycin + penicillin; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 1:1 Dulbecco's modified Eagle's medium (DMEM)/F12 (Hyclone; GE Healthcare Life Sciences) in the volume fraction.

Dulbecco's modified Eagle's medium [(DMEM)/F12] was obtained from GE Healthcare Life Sciences (Logan, UT, USA). Mouse anti-MMP-2 monoclonal antibody (cat. no. SC-13595) and mouse MMP-9 monoclonal antibody (cat. no. SC-21733) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal phospho-AKT^S473 antibody (cat. no. #4060) and mouse AKT monoclonal antibody (cat. no. #2920) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal β-actin antibody (cat. no. BS-0061R) was purchased from Bioss (Beijing, China). Biotin-labelled goat anti-rabbit secondary antibody (cat. no. ZDR-5306) and biotin-labelled goat anti-mouse secondary antibody (cat. no. ZDR-5307) were purchased from ZSJB-BIO (Beijing, China). Recombinant human sonic hedgehog N-terminal peptide (rhSHH; cat. no. 1314-SH) was purchased from Toronto Research Chemicals (Toronto, Canada). Cyclopamine (cat. no. BML-GR334) and GM6001 (cat. no. BML-EI300), were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Ly294002 (cat. no. L9908) was from purchased from Sigma-Aldrich (St. Louis, MO, USA)