FIG. 5.

Proliferative properties of fibroblasts lacking both Cited2 and INK4a/ARF. (A and B) Fibroblasts were harvested simultaneously from independent embryos at 13.5 dpc. Cumulative fibroblast growth is shown as a plot of CPD versus passage number. Each line represents fibroblasts isolated from an independent embryo. Fibroblast genotypes (w, wild-type; n, null) for Cited2 and INK4a/ARF alleles are indicated in panel B. (C) Proliferative capacity of fibroblasts at passage 4. Fibroblasts were plated in quadruplicate into 12-well plates (2.5 × 10⁴ cells per well) and fixed at the indicated time points, and relative cell numbers were determined by using crystal violet as previously described (25). Data (means ± SEMs) were normalized for cell numbers at day 1. The genotypes are indicated in panel B. (D) Colony formation assay. Fibroblasts (passage 1) were plated at a density of 4,000 cells per 9-cm plate, and colonies were visualized with Giemsa stain after 7 days. Fibroblast genotypes are indicated as wild-type (w) or null (n) for Cited2 and INK4a/ARF alleles, as indicated in panel B.