Fig. 6. Time course of caspase activation and cleavage of the caspase substrate poly(ADP-ribosyl) polymerase (PARP) in differentiating cells. Western blot analysis of caspase-3 activation (A) and PARP cleavage (B) in human papillomavirus (HPV) 31-positive human foreskin keratinocytes (HFKs) and normal HFKs induced to differentiate in methylcellulose. Hr, hours.

Fig. 7. Assessment of caspase activation and apoptotic regulators upon calcium-induced differentiation. (A) HPV-31-positive HFKs or normal HFKs were exposed to high calcium for up to 96 h, and cell lysates were analyzed for caspase activation by Western blot analysis. (B) Cells were treated as in A and analyzed by Western blot analysis for Bcl-2, Bax, and survivin levels.

Fig. 8. Analysis of enzymatic caspase-3/7 activity in HPV-31-positive HFKs induced to differentiate in high calcium. Caspase activity was measured 96 h postexposure to high-calcium medium containing DMSO alone or 20 mM the general caspase inhibitor Z-VAD-FMK or the caspase-3 inhibitor Z-DMQD-FMK. Caspase activation was measured by using fluorometric analysis of caspase substrates. Shown are the relative fluorescence units after correcting for background levels in undifferentiated cells. ST, staurosporine.

Fig. 9. Cleavage of E1 in vitro by caspase-7 and in vivo upon staurosporine-induced apoptosis. (A) GST-E1 (amino acids 1-170) and GST-E1D49A were incubated with increasing concentrations of recombinant caspase-7 (C7) in the absence or presence of Z-VAD-FMK (Ci). Cleavage reactions were analyzed by immunoblotting by using an antibody to GST. (B) Western blot analysis was performed on lysates from C33A cells expressing full-length YFP-tagged E1 or YFP-tagged E1D49A that were left untreated (UT) or incubated with 0.5 or 0.8 mM staurosporine for 4 h. The primary antibodies used were to GFP, the cleaved forms of caspase-3 and -7, and GAPDH.