Figure 2

Effects of HPV-16 sequences on the steady state level of GUS mRNA and GUS protein. a) Schematic representation of the HRSp-GUS retroviral reporter vector [26,27] and the location of the GUS northern probes and the Real Time PCR amplicon. b) Northern Blot analysis of mRNA from SiHa cell pools (only GUS probe 1 experiment is shown). Blotting of mRNA from the HaCaT cell pools is not shown, but yielded similar results. c) GUS mRNA steady state levels. For each construct the steady state levels of the GUS mRNA and PURO mRNA were determined with real-time RT-PCR. The GUS mRNA levels were normalized with the PURO mRNA. The data plotted is the average of results from three independent pools of infected SiHa or HaCaT cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean. d) Reporter protein produced per reporter mRNA. For each construct the steady state GUS activity and total protein levels were determined. The GUS activities were normalized with the total protein levels. Protein/RNA ratios of the different reporter mRNA was evaluated by calculation of the ratio between the steady state levels of GUS protein to GUS mRNAs (not normalized for PURO) The data plotted is the average of results from three independent pools of infected SiHa or HaCaT cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean. e) Cellular distribution of Reporter mRNAs. The ratio of cytoplasmic to nuclear RNA for the GUS mRNA (left), PURO mRNA (middle) and GAPDH mRNA (right) in the different reporter cell lines.