Fig. 6.
Low levels of ectopic Orc1 induced caspase-3 activity. (A) HeLa cells were transfected with 1 µg of either pCI, pFHsOrc1 (●) or pFHsOrc2 (○). At the indicated times post-transfection, extracts from ~106 cells were prepared by the freeze-thaw method and assayed for Z-VAD-fmk-sensitive caspase-3 activity using the colorimetric CaspACE™ Assay System (Promega). Activities observed with pCI were subtracted from those observed with Orc expression vectors. The fraction of unattached transfected cells is indicated by broken line (0.5=50%). (B) CHO cells transfected with pCI, pFCgOrc1 (●) or pFCgOrc2 (○) were analyzed as in panel A. (C) Amounts of Orc1 (◆) and Orc2 (◇) proteins in CHO cells transfected with pFCgOrc1 were determined by densitometry of data such as those in panel E. Comparisons were made only where images were not saturating. These results were compared with the appearance of caspase-3 activity (●) at the beginning of transfection. Comparison with standard amounts of recombinant Orc protein fractionated in parallel was used to determine the cellular concentration of Orc. (D) Amounts of Orc1 (◆) and Orc2 (◇) in CHO cells transfected with pCgOrc2 were determined from data such as those in panel E, and then compared with caspase-3 activity (○) at the beginning of transfection. (E) Total CgOrc1 (Orc1) and CgOrc2 (Orc2) in CHO cells following transfection with pFCgOrc1 was detected by immunoblotting with both anti-CgOrc1 and anti-CgOrc2 IgG. Two independent experiments are shown. In the second, different exposures of the same gel were required to visualize both Orc1 and Orc2. (F) Total CgOrc1 and CgOrc2 in CHO cells following transfection with pFCgOrc2 was determined as in panel E. Two independent experiments are shown.