Figure 2.

Apoptin inhibits mitotic cells from progressing through anaphase and telophase. U2OS and Saos-2 cells were transiently transfected with plasmid-encoding flag-SPRR4 or flag-apoptin, and processed for immune fluorescence analysis as described in the legend of Figure 1. Both flag-apoptin-transfected and untransfected mitotic cells were scored according to their phase in mitosis. Representative images of the four phases in U2OS are in (a) from left to right prophase, metaphase, anaphase and telophase. Apoptin-positive mitotic U2OS cells could be found in prophase (b), and metaphase (c), but were scarce in anaphase and telophase, whereas SPRR4-positive mitotic U2OS cells were readily found in anaphase (d), and telophase/cytokinesis (e). Apoptin transfected mitotic cells were scored as described above, grouped as prophase/metaphase and anaphase/telophase and compared to untransfected cells in the same experiment. The results from three separate experiments (represented as means±S.D.) in which 100 mitotic cells per group were counted are given. Panel (f): Saos-2 cells (**P=0.0041). Panel (g): U2OS cells (**P=0.0055). The distribution of ‘pro/meta' and ‘ana/telo' phases did not significantly differ between apoptin-positive p53-wt U2OS and p53-null Saos-2 cells (P=0.38 in a paired two-tailed t-test). Scale bar in panels (a–e): 5 μm. Panels (h–j): apoptin-positive cells are shown that undergo cell death during mitosis: (h) after spindle formation, (i) during abnormal cytokinesis, and (j) during normal cytokinesis. Note membrane blebbing and DNA fragmentation in the dying cells. Scale bar: 10 μm