Figure 3.

Schematic representation of the experimental procedure. The first day, two batches of H1299 cells are concomitantly transfected with plasmids encoding Rluc-p53 and eGFP along with a plasmid encoding HPV16 E6 or with empty vector. The second day, cells from both batches are reseeded in 96-well white plates at low density (3 × 10³ cells/well) as described in the text. In experiments testing candidate inhibitors, at 8 hours post-reseeding test compounds are added to both Batch 1 and Batch 2 samples. At day three, fluorimetric and luminometric measurements are performed. The RLU/FU ratio of cells cotransfected with empty vector (Batch 1) is set as 100% and related to the RLU/FU ratio of cells cotransfected with the E6-expressing plasmid (Batch 2) in order to calculate the ratio of Rluc-p53 degradation mediated by E6.