Figure 4. Analyses of Fibroblasts Lacking All A- and E-type Cyclins.

(A) Diagram illustrating generation of quadruple knockout fibroblasts via tetraploid complementation. Right-hand panel shows Western blot analysis of cyclin A2 levels in A1^-/-A2^f/fE1^-/-E2^-/- cells transduced with adenovirus encoding empty vector (Control) or Cre (A1^-/-A2^Δ/ΔE1^-/-E2^-/-). (B) Incorporation of [³H]-thymidine and (C) the percentage of BrdU-positive cells in A1^-/-A2^f/fE1^-/-E2^-/- fibroblasts transduced as above. Shown are mean values ± SD. (D) Upper panel: fibroblasts, cultured in medium containing 10% serum, were stained with propidium iodide and analyzed by FACS. Lower panel: cells were placed in serum-free medium for 3 days, stained with propidium iodide and analyzed by FACS. (E) In vitro proliferation of fibroblasts. Equal numbers of cells were plated at the beginning of the experiment. Cells were counted every day for 7 days. This experiment was performed using cells immortalized with dominant-negative p53, as cyclin E-deficient cells undergo premature senescence in culture (Geng et al., 2003). (F) The levels of the indicated proteins in cyclin A- and E-deficient fibroblasts, detected by Western blotting. (G) cyclin B1, Cdk1 or Cdk2 were immunoprecipitated from protein lysates, and used for in vitro kinase reactions with histone H1 as a substrate.