FIG. 2.
The level of E6 expression affects both the level of endogenous procaspase 8 and the cellular response to TNF. (A) The level of cellular procaspase 8 changes with the amount of E6 expressed. Control cells (U2OSE6tetC) and cells in which the level of E6 could be regulated by the amount of doxycycline (DOX) present in the media (U2OSE6tet24) were cultured in the presence of the indicated concentration of doxycycline and then lysed and analyzed for their levels of procaspase 8 expression by immunoblotting (top). The middle blot analyzes β-actin expression as a loading control. The level of HA-E6 expressed was also analyzed by immunoprecipitation (2 × 106 cells per preparation) using anti-HA monoclonal antibodies and then detected by using anti-HA rat polyclonal antibodies conjugated to HRP (bottom). To obtain the graphical results, immunoblots were performed for three separate experiments and then analyzed using the Odyssey Infrared Imaging System. The error bars represent the standard deviations. (B) The level of E6 expression affects cell viability in the presence of TNF. U2OS cells or U2OSE6tet24 cells, grown in the presence of the indicated levels of doxycycline, were treated with 2, 5, or 10 ng/ml TNF in the presence of cycloheximide (5 μg/ml). Following 16 h of incubation, cell viability was determined by the MTT assay and is presented as a percentage of viable cells (with untreated cells serving as the control). Error bars represent standard deviations. *, 0.95 level of confidence; **, 0.99 level of confidence.