Fig. 4

E7-mediated rapamycin resistance requires an intact LxCxE motif. a DNA synthesis in organotypic culture of keratinocytes expressing HPV-16 E7 mutants was detected by BrdU staining. Arrows indicate positive nuclei. b The percentage of BrdU-positive/total nuclei was determined by direct nuclei counting. The bars correspond to values obtained in at least three independent experiments. (*) p < 0.05. c Growth curve of monolayer cultures of keratinocytes treated with 100 ng/ml rapamycin for 7 days. Results are representative of three independent experiments performed in triplicates. (*) p < 0.05; (**) p < 0.001. d Alamar Blue cell proliferation assay was used to evaluate the effect of rapamycin on the proliferation of PHK silenced for pRb expression HPV16 E7 transduced PHKs. Gene silencing was performed as described in Fig. 2. Experiments were carried out in triplicate for each time point. Scale bars, 20 μm