FIG. 1.

The expression of procaspase 8 in cells is linked to caspase 8 activity and to TNF sensitivity and may be influenced by HPV-16 E6. (A) U2OS cells stably transfected with pHA-E6S express various amounts of procaspase 8. Cells (1 × 10⁶) were lysed in 200 μl Laemmli buffer, and 20 μg (total protein) of the lysate was separated by SDS-PAGE. Procaspase 8 was detected by immunoblotting using anti-caspase 8 antibodies (top), and loading was normalized by immunoblotting using anti-β-actin antibodies (middle). The level of HA-E6 expressed in each of these cell lines 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) Caspase 8 activity measurements correlate well with estimates of protein levels. The indicated cell lines were treated with TNF (5 ng/ml) in the presence of cycloheximide (5 μg/ml) for 6 h and lysed. The level of caspase 8 activity was then measured using IETD-AMC as the substrate in the presence and absence of the caspase 8 inhibitor Ac-IETD-CHO. The activity in wells containing the inhibitor was subtracted from that in wells lacking the inhibitor, following normalization for the amount of protein in each cell lysate. Error bars represent standard deviations. (C) HA-E6 affects the cellular response to TNF. Each of the indicated cell lines was treated with TNF (5 ng/ml) in the presence of cycloheximide (5 μg/ml). After 16 h, cell viability was determined by the MTT assay and is presented as a percentage of viable cells, with cells untreated with TNF serving as the control. *, 0.95 level of confidence; **, 0.99 level of confidence.