Figure 4.

ARF-induced arrest of triple knock out (TKO) cells does not depend on p21^Cip1 and is not overridden by HPV E7. (A) TKO mouse embryo fibroblasts (MEFs) infected with retroviruses encoding CD8 or ARF (96 hours postinfection) or wild-type p53 (48 hours postinfection) were lysed, and separated proteins were blotted with antibodies to p53, p21^Cip1, and ARF. (B) TKO MEFs infected with retroviruses encoding E7 or CD8 were lysed 48 hours after infection, and separated proteins were blotted with antibody to E7. (C) TKO MEFs infected with MSCV-GFP-IRES or MSCV-GFP-IRES-E7 retroviruses were re-plated 48 hours later and infected with retroviruses encoding CD8, ARF, or p19^INK4d. Infected cells were analyzed 96 hours postinfection using an FITC filter to detect GFP-IRES-E7 infected cells (green), immunofluorescence to score 5-bromodeoxyuridine incorporation (red), and antibody to p19^ARF followed by Alexa 350 stain to visualize nucleolar ARF (blue). Alternatively, p19^INK4d was detected using a rabbit anti-p19^INK4d antibody followed by biotinylated anti-rabbit immunoglobulin and streptavidin Texas red to ensure >90% infection efficiency of TKO and E7-containing TKO cells.