FIGURE 3.

N1 antiapoptotic activity is strictly p53-dependent. PG13-luciferase (a), p21^waf-1-luciferase (b), or p53 promoter-luciferase (pp53) (c) reporter constructs were transiently transfected in HEK293 cells together with β-galactosidase-expressing vector, as detailed under “Materials and Methods.” Twenty-four h after transfection, cells were treated for 16 h with recombinant N1 (1 μm) or an equivalent volume of control supernatant (KG). The luciferase and β-galactosidase activities were measured as detailed under “Materials and Methods.” Bars correspond to the ratio of luciferase/β-galactosidase and show the means ± S.E. of 3–6 independent experiments performed in triplicate. d, cells treated with recombinant N1 (1 μm) or KG for 8 h were analyzed for levels of p53 mRNA by real time PCR as indicated under “Materials and Methods.” Bars correspond to p53 mRNA levels expressed as a percentage of control-treated cells and are the means ± S.E. of three independent experiments carried out in duplicate. e, recombinant N1 (1 μm) or equivalent volume of KG were applied to p19^arf−/− and p19^arf−/−p53^−/− fibroblasts. After 4 h of incubation, cells were treated with N1 or KG before being challenged with staurosporine (1 μm, 2 h) and processed for caspase-3 activity measurement. Bars, means ± S.E. of five independent experiments performed in duplicate. p19^arf−/− and p19^arf−/−p53^−/− cells (f), seeded on glass coverslips, were treated as in e and then processed for TUNEL labeling as described under “Materials and Methods.” f, representative pictures of TUNEL labeling of control (KG) and N1-treated p19^arf−/− cells. g, TUNEL-positive nuclei were counted for 10 independent optical fields. Bars, means ± S.E. of labeled nuclei/field. *, p < 0.05; **, p < 0.001; ***, p < 0.0001.