FIG. 4.
Defective senescence of Rb1ΔL/ΔL mutants contributes to immortalization. (A) MEFs were subjected to a 3T3 culture protocol to induce entry into senescence. We measured the number of passages that it took the cells to senesce and the number of passages it took them to become immortalized. Senescence was defined as the first passage without a population doubling, and immortalization was the next passage where cells resumed doubling and continued to double each passage thereafter. Scatterplots showing the passage where each wild-type or Rb1ΔL/ΔL mutant culture ceased to proliferate are shown at the left. Plots that reveal when cultures resumed proliferating are shown at the right. Horizontal bars represent the mean of each measurement. P values are 0.66 (for entry) and 0.04 (for escape). (B) H&E and immunohistochemical staining of liver sections from wild-type and Rb1ΔL/ΔL mutant mice stained with Ki67 antibodies (or immunoglobulin G [IgG] control). Each field of view is centered on a portal duct to ensure equivalent orientation of the tissue. (C) DNA content of nuclei extracted from livers was analyzed by propidium iodide staining and flow cytometry. Each ploidy content category is expressed as a percentage of the total number of nuclei analyzed. Error bars indicate 1 standard deviation from the mean of at least three replicates. (D) Protein expression of known E2F target genes, as well as other pRB family proteins, is shown for nuclear extracts prepared from hepatocytes.