Cell culture, transfection and promoter activity determination

Bm12 cells at logarithmic growth phase were used for transfection. Plasmid DNAs were mixed with Lipfectamine 2000 (Invitrogen) and added to cells in each well of 12-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-drived vectors containing tested promoters. After 6-h post transfection, the old medium was replaced with fresh Grace medium containing 10% FBS. The cells were cultured for additional 48 h at 28 °C before promoter activity assay. The cells were washed once with filtered PBS and then lysed in 200 μL Passive Lysis Buffer (Promega). Luciferase activity of the supernatant was analyzed using the Dual-Luciferase Assay System (Promega) according to the manufacturer’s instruction with a luminometer (IBA7300, Veritas, Turner Biosystems). All assays were conducted three times.