Luciferase-Based Assay for HPV-31 Replication

The luciferase-based replication assays of the C33a cells were essentially conducted as previously described [33 (link)], with a slight modification. Briefly, 80 ng each of the HPV-31 E1, E2, and pFLORI31 constructs was co-transfected along with Renilla luciferase at 16 ng per well on a 12-well plate. Twenty-four h post-transfection, the cells were exposed to 2 mM caffeine (dissolved in PBS; Sigma-Aldrich, St. Louis, USA) for the indicated times. The cells were lysed in Promega Dual-Glo luciferase reagent, and both the firefly and Renilla luciferase activity were determined using PHERAStar FS (BMG Labtech, Ortenberg, Germany). The firefly luciferase activity was normalized to the Renilla luciferase activity, and the value for the control was set as 100%.