Penicillin and streptomycin sulfate

Human oral squamous cell carcinoma cell lines SCC9, SCC15, SCC25 and CAL27 were obtained from the American Type Culture Collection. Human oral keratinocyte (HOK) cells were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. SCC15, SCC25, CAL27 and HOK cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin sulfate (all Gibco; Thermo Fisher Scientific, Inc.). SCC9 cells were cultured in 1:1 DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS, 1% penicillin and streptomycin sulfate, 1% sodium pyruvate (Gibco; Thermo Fisher Scientific, Inc.) and 400 ng/ml hydrocortisone (MedChemExpress). All cells were cultured at 37°C and 5% CO₂.

After the BM-EPCs were transfected with rAAV₆-GFP and rAAV₆-CXCR4, they were cultured at 37˚C with 5% CO₂ in a humidified atmosphere with Endothelial Cell Growth Medium-2 (Lonza Group, Inc.), supplemented with 10% FBS, 100 U/ml penicillin and streptomycin sulfate (Thermo Fisher Scientific, Inc.), and SDF-1 at 20 µg/l for conventional culture. Initially, the BM-EPCs in the normoxia group were cultured under normoxic conditions (5% CO₂, 95% air atmosphere). BM-EPCs in the hypoxia group were cultured under anaerobic conditions (5% CO₂, 95% N₂) free of FBS for 4 h, and then cultured under normoxic conditions with PBS for 6 h. The BM-EPCs were subsequently divided into four groups: Normoxia + Sham (Empty vector) group, Normoxia + CXCR4 group, Hypoxia + Sham group, and Hypoxia + CXCR4 group. In additional experiments, the BM-EPCs were divided into these four groups: Hypoxia + Sham group, Hypoxia + CXCR4 group, Hypoxia + CXCR4 + PI3K inhibitor (LY294002, 20 µmol/l for 1 h) group, and Hypoxia + CXCR4 + CXCR4 inhibitor (AMD3100) group.