Loss of Robustness and Addiction to IGF1 during Early Keratinocyte Transformation by Human Papilloma Virus 16
Figure 3
P53 and Rb under basal conditions and during starvation.
A. Representative western blots of whole cell lysates of primary keratinocytes, early and late HF1 cells under basal growth conditions. Blots show the status of HPV16 E7 protein, Rb and p53. Graphs show quantification of these blots. Protein densities were normalized to the densities of the bands in early HF1 cells. Error bars represent the standard deviations of three independent experiments. Tubulin serves as a loading control. B. Real-time PCR for NOXA expression under basal conditions. RNA was isolated from untreated cells and subjected to reverse-transcription followed by real-time PCR with specific primers for NOXA and HPRT as control. NOXA results were normalized to the HPRT results. Error bars represent the standard errors of three independent experiments. C. Real-time PCR for NOXA expression in starved vs. untreated cells. Cells were serum starved for 24 h, RNA was isolated, reverse-transcribed and subjected to real-time PCR as in B. Fold induction represents results of starved cells vs. non-starved cells. Error bars represent the standard error of three independent experiments. D. Reporter assay for p53 activity in untreated and serum starved cells. Cells were transfected with firefly-luciferase (FFL), either under the control of 17 p53-response elements, or under the control of mutant response elements as control. For transfection control cells were co-transfected with renilla-luciferase under the control of CMV promoter. 24 h after transfection cell medium was replaced with normal growth medium or starvation medium, for another 24 h. Cells were lysed and subjected to luminisence assay. Error bars represent standard errors of four independent experiments.