Overexpression of Spp1 in Schwann cells

SCs were cultured in DMEM (GIBCO, Grand Island, NY) with 100 IU/mL penicillin, 10% fetal calf serum, and 100 g/mL streptomycin at 37 °C and 5% CO₂. The SCs were identified by examining the immunofluorescence of an antibody to the marker S100, and the final cells were found to comprise 98% SCs. The Spp1 overexpression plasmid pcDNA3.1-Spp1 was constructed as previously described [21]. A mixture of pcDNA3.1-Spp1 plasmid and X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany), or X-treme GENE HP DNA Transfection Reagent and an empty vector were then transfected into SCs for 48 h. After that, real-time quantitative (q)PCR and Western blot analyses were conducted. The pcDNA3.1-Spp1 overexpression experiments were repeated three times.