Effects of FGF2 on SDF-1 promoter activity. (A) Schematic representation of the SDF-1 promoter-reporter construct pGL3.0+2071bp. An approximately 2.2-kb DNA fragment containing SDF-1 upstream sequences and the partial coding sequence (−2030 to +149 relative to the transcription start site) was amplified by PCR, and the fragment (−2030 to +41) was inserted into the reporter vector pGL3.0-Basic. (B) MS-5 cells were transfected with pGL3.0-Basic (250 ng/well) plus phRL-SV40 (Renilla luciferase reference control plasmid, 2.5 ng/well) or with pGL3.0+2071 bp (250 ng/well) plus phRL-SV40 (2.5 ng/well). All assays were carried out at least in triplicate sets and repeated twice. After 24-hour incubation, firefly luciferase activity was evaluated. Transfection efficiency was adjusted by Renilla luciferase activities (relative luciferase activity). CPS indicates counts per second. (C-D) pGL3.0+2071 bp-transfected MS-5 cells were stimulated with or without FGF2 (50 ng/mL) for a period of 72, 96, or 120 hours. Cell lysates and culture supernatants were obtained for luciferase assay and SDF-1 ELISA, respectively. All experiments were performed at least in triplicate and repeated 3 times. The percent relative luciferase activity was defined as follows: (relative luciferase activity in FGF2-stimulated group/relative luciferase activity in control group) × 100 (%). The percent SDF-1 secretion was calculated as follows: (SDF-1 secretion in FGF2-stimulated group/SDF-1 secretion in control group) × 100 (%). Error bars denote standard deviations of 3 experiments; asterisks denote statistical significance (*P < .05; **P < .001).