Ng 108 cells

Bone marrow stem cells (Cyagen Biosciences Inc., USA), NG-108 cells (ATCC, USA), fetal bovine serum (Gibco, USA), Dulbecco’s Modified Eagle Medium (DMEM)/F12 Culture medium (Gibco), trypsin (Sigma, USA), penicillin (Gibco), streptomycin (Gibco), beta-mercaptoethanol (Amresco, USA), all trans-retinoic acid (Sigma), forskolin (Peprotech, UK), basic fibroblast growth factor (bFGF) (Peprotech), platelet-derived growth factor (PDGF) (Peprotech), recombinant human heregulin-b1 (heregulin-b1) (Peprotech), S-100 antibodies (Abcam, USA), glial fibrillary acidic protein (GFAP) antibodies (Abcam), β-actin antibody (Proteintech Group, USA), RIPA cell lysate (Biotime Company, CHN), BCA protein determination reagent (Biotime Company), SDS PAGE gel preparation kit (Biotime Company), DAB light liquid (Biotime Company), goat anti-rabbit IgG2 (Biotime Company), and Tris-buffered saline plus Tween 20 (TBST) (Biotime Company) were all used for the current study. Annexin V-FITC/PI apoptosis and cell cycle kit(Liankebio company,CHN),Mitochondria Staining Kit(JC-1) (Liankebio company).

Total proteins were extracted from HeLa (ATCC) and NG-108 cells (ATCC). Briefly, collected cells were lysed with RIPA lysis buffer (10 mM Tris-HCl pH 8.0, 140 mM sodium chloride, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid, and 0.5 mM ethylene glycol tetraacetic acid) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and the protein amount was measured (Pierce bicinchoninic acid protein assay kit, Thermo Fisher Scientific, Rockford, IL). Equal amount (30 µg) of protein samples were fractionated by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. After incubation with 5% (wt/vol) nonfat milk in TBST (10 mM Tris. pH 8.0, 150 mM NaCl, and 0.5% (vol/vol) Tween 20) for 1 h, membranes were incubated with various primary antibodies (see Supplementary Information Table 1) overnight at 4 °C. Alpha-tubulin or actin was used as loading control. Membranes were washed three times with TBST for 15 min followed by probing with secondary antibody of goat anti-rabbit or goat anti-mouse antibody. Blots were washed three times for 15 min with TBST and developed by using the LiCor system (LiCor CLx). Expression bands were analyzed by Image Studio Lite (LiCor 5.2.5) according to the manufacturer’s manual.