Analysis of the inhibition of U2OS cancer cell growth. We added recombinant adenovirus at the concentration of 8.8 × 105 pfu/ml to the medium. (a) Numbers of U2OS cells after infection of each virus construct. Cell numbers at every 2 days starting from 1 day after infection are shown. Cell number at 1 day after infection is indicated as 1.0. (b) Telomere digestion by expressed chimeric endonucleases. We performed TRF assay with U2OS cells 3, 7 and 13 days after viral infection. (c) Immunoblot analysis of U2OS cell extracts harvested 3, 7 and 13 days after viral infections. p53 and p16 are markers of cellular senescence. Anti-HA antibody (HA) was used to detect chimeric endonucleases. α-tubulin and β-actin expression were used as loading controls. (d, e) We performed a senescence-associated (SA) β-galactosidase assay (see Methods) on day 13 cells. EN-T expressing cells show senescent phenotype, that is, an enlarged and flattened appearance and increased SA β-galactosidase activity as shown in (d) (two different fields are shown for each sample; the scale bar is 100 μm), and the percentage of the SA β-galactosidase positive cells is shown in (e) (bar: 95% confidence interval).