Figure 5.

Growth inhibition of dense cultures by high levels of p27^kip1. p27-47 cells were grown to confluency in medium containing 10% serum and incubated overnight in the presence or absence of 1 mM IPTG. (A) Cells were either harvested at this time (quiescent, IPTG) or refed with medium containing 10 ng/ml PDGF and 10% calf serum (CS); cultures pretreated with IPTG received fresh IPTG. Cells were harvested 24 h later for analysis of cell cycle position by flow cytometry. (B) Cultures were treated as in A. Cell extracts were immunoblotted with antibody to p27^kip1 (top panel) or cyclin A (middle panel) or were immunoprecipitated with antibody to cyclin A for determination of cyclin A/cdk2 activity using histone H1 as substrate (bottom panel). (C) Confluent cultures were pretreated with IPTG overnight and stimulated with PDGF and serum for 24 h. Cultures were trypsinized and replated at a density of 700 cells/cm² in medium containing 10% serum. Cells were cultured for 7 d in the presence or absence of 1 mM IPTG and were photographed at a magnification of 100×.