Figure 5.

Immortality and partially transformed phenotype of TKO MEF populations. (A) Cultures derived from individual embryos were cultivated according to the 3T3 protocol. The graph shows the accumulated number of doublings that representative cultures have undergone. Full legend is as indicated in the graph. Groups of MEFs with the same genotypes or phenotypes are also indicated (CTRL, controls: wt, Rb^−/− and p107^−/−;p130^−/−). (B) Western blot analysis of p53 and of p16^INK4A, p19^ARF, and p21^CIP1 cell cycle inhibitors in early and late passage TKO clones. TKO cells (1–5) extracts were prepared at different passages, as indicated. Equal quantities of proteins were analyzed. (C) Proliferation of TKO and control cells after plating at low density. 1.3 × 10³ cells of each genotype were plated per 10-cm plate, and after 2 wk, colonies were stained and counted. At least five experiments were performed; average numbers and standard deviations (error bars) are shown. (D) TKO cells were infected with an empty retrovirus (pMIG) or with the same retrovirus expressing a human RB cDNA (pMIG-RB) and then plated at low density. The plates were stained with Giemsa to visualize the colonies 2 wk after plating. (E) Representative examples of TKO and control MEFs grown for 2 wk at confluency. TKO cells form a very dense layer with foci growing on top of it and with a tendency to peel off the plate. This phenomenon was not observed with wt, Rb^−/−, p107^−/−;p130^−/−, and p53^−/− cells. (F) Ability of MEFs with different genotypes to form colonies in soft agar. Pictures were taken at the same magnification (bar, 400 μm) after 24 d in culture.