Comparison of HPV-5 and HPV-16 LCR transcriptional activity in four different cell lines. A pBlue-Topo vector (Invitrogen) containing the HPV-5 and HPV-16 LCR were transfected into HaCaT, C33A, NIKS and W12E cells to compare their ability to activate transcription of a beta-galactosidase reporter gene. The ability to transfect all cell lines was optimized by using a CMV promoter cloned in the same vector as the HPV LCR constructs. One day prior to transfection all cells were seeded in 24-well plates, NIKS and W12E cells were without feeder cells. All cells were transfected with plasmid DNA by FuGENE 6.0 (Roche) according to the manufacturer and protein expression was measured 3 days later. For detection the transfected cells were fixed with 1–2% formaldehyd in PBS for 5 minutes, incubated in dark with X-Gal staining buffer (2 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6 and 1 mg/ml X-Gal in PBS) in room temperature up to 24 hours. Cells were washed with 5% dimethylsulfoxide in PBS and the number of blue cells were visualised and counted using a light microscope. Results were expressed as percentage of blue cells of the HPV-16 LCR transfection, which was set to 100%. Data are means +/- standard errors of the means (n = 4).