Figure 4.

Cdc6 is required for efficient cyclin D expression and G₁-progression. (A) Quiescent hTERT-expressing HDF were transfected with siCdc6 or siCon oligonucleotides, then stimulated with serum. After 0 hr, 4 hr, 14 hr and 16 hr of serum stimulation soluble fractions were prepared and analyzed for expression of various proteins using SDS-PAGE and immunoblotting (upper). In a parallel experiment, siCdc6- or siCon-transfected cells were stimulated with serum for 20 hr, then analyzed by flow cytometry (lower). (B) Quiescent HDF were infected with adenovirus encoding HA-tagged Cdc6-A4 or with an empty-vector control adenovirus (AdCon). 24 hours after infection, cells were stimulated with serum. At different times after serum stimulation cells were harvested for PI staining and FACS analysis to determine cell cycle distributions. (C) Quiescent HDF were infected with Ad HA-Cdc6-A4 or AdCon. 24 hours after infection, cells were stimulated with serum. After 0 hr, 4 hr, 12 hr and 16 hr of serum stimulation soluble proteins were extracted and analyzed by SDS-PAGE and immunoblotting for expression of various proteins as indicated. (D) Quiescent HDF were infected with Ad HA-Cdc6-A4 or with an empty-vector control adenovirus (AdCon). 24 hours after infection, cells were stimulated with serum for 4 hr. Lysates from the resulting cells were immunoprecipitated with anti-cyclin D1 antibodies. Immune complexes were assayed for Rb-directed kinase activity in vitro.