Cdc25A^128Gln alters the growth characteristics of mouse embryo fibroblasts (MEFs).

MEF cultures were established from C57BL/6J embryos (Cdc25A^128His) and the congenic strain, B6.C-H7^b/By, embryos (Cdc25A^128Gln). (A) Growth analysis of MEF cultures. Initially, 1 × 10³ cells were plated out. At the indicated times, cells were counted. Procedures at each time point were performed in duplicate. The graph is representative of 3 independent experiments, each using independently derived MEF cultures. Data are averages ± SD. (B) Analysis of S-phase progression in MEF cultures. Cells were grown to confluence and starved at low serum for 24 hours to synchronize the cells in G0/G1. Cells were then split into new media containing serum and grown for the indicated times. Cells were harvested, fixed, and stained with PI. Stained cells were analyzed by flow cytometry, and the percentage of cells in S phase was calculated by using the Multicycle (Phoenix Flow Systems) program. The graph is representative of 3 independent experiments using 3 independently derived MEF cultures for each allele. (C) Analysis of CDK2 H1 kinase activity in MEF cultures. Cells were grown in low-serum media to synchronize cells in G0/G1. Cells were then transferred to new media containing serum and grown for the indicated times. The cells were lysed, and CDK2 was immunoprecipitated. Histone H1 kinase activity was measured.13 14 The gel is representative of 2 independent experiments using 2 different independently derived MEF cultures for each allele.