Figure 5. rDNA promoter activity and level of UBF phosphorylation upon E7 and/or p14^ARF depletion in the HPV16 positive CaSki cervical carcinoma line.

E7 and/or p14^ARF were depleted in the HPV16 CaSki cervical carcinoma cell line using siRNA and Lipofectamine 2000 reagent. CaSki cells were transiently transfected with the pHrDNA-IRES-Luc plasmid along with the Renilla internal control pRLTK, siRNA control (Si CTR), E7 siRNA and/or p14^ARF siRNA and lysed at 48 h. (A) Western blot analysis of CaSki cells extracts probed with antibodies to phosphorylated UBF (P-UBF Ser 388), p14^ARF and actin (as a loading control). Quantification of western blots was done by measuring the relative intensity of the bands compared to internal controls (Actin), the values given are in arbitrary units. (B) Expression of E7 mRNA assessed by quantitative real time RT-PCR. (C) rDNA promoter activity. Reporter activity was calculated as the ratio of firefly luciferase activity to reference Renilla luciferase activity, and normalized so that that luciferase activity in control vector transfected cells equaled 1. The data are representative of three independent experiments and are the means of triplicate samples +/− SD (**p<0.01).