FIG. 7.

Senescent phenotype induced by E2F1 is p14^ARF dependent. (A) U2OS cells were infected with control (B0)-, wild-type E2F1 (B-WT)-, transactivation defective CterD1 E2F1 (B-CterD1)-, or p14^ARF (B-ARF)-expressing retroviruses and selected in puromycin. The cells were counted or labeled with [³H]thymidine for 3 days to determine the percentage that synthesized DNA (%LN) prior to infection (Preinfect), prior to selection (Preselect), or 2 to 3 days after selection (Postselect). In parallel, the cells were stained for SA-β-Gal activity prior to infection (Preinfect) and after selection (Postselect). For each determination of percent labeled nuclei and SA-β-Gal activity, at least 400 cells were counted from two independent culture dishes. (B) A375 cells were infected and analyzed for cell number, percent labeled nuclei, and SA-β-Gal activity, as described for U2OS cells (A). (C) Protein lysates were prepared from postselected U2OS cells infected with control (B0)-, wild-type E2F1 (B-WT)-, CterDl E2F1 (B-CterDl)-, or p14^ARF (B-ARF)-expressing retroviruses and analyzed by Western blotting for p53, p21, E2F1, p14^ARF, and QM (control) protein.