FIG 6.

Discrete defects in E2F transcriptional control in Rb1^ΔG/ΔG MEFs. (A) The relative expression levels of six pRB-regulated E2F cell cycle target genes are shown. Each transcript was quantified from serum-starved Rb1^+/+, Rb1^ΔG/ΔG, and Rb1^−/− fibroblasts with the relative level of message present in wild type scaled to 1. (B) Serum-starved cell cultures were pulse-labeled and stained for BrdU incorporation along with total DNA using propidium iodide. The proportion of cells in each respective cell cycle phase was determined by flow cytometry. (C) Microarray analysis was performed on serum-starved Rb1^+/+, Rb1^ΔG/ΔG, and Rb1^−/− cells, and E2F target gene expression is shown. Log ratios of expression of Rb1^ΔG/ΔG or Rb1^−/− relative to that of wild-type control are shown as a heat map, and genes were clustered based on similarity of expression. The genotypes of each lane are shown above. Categories of E2F target genes are shown to the right. (D) Serum-starved cells of the indicated genotypes were stimulated to reenter the cell cycle with 10% FBS. Rb1^+/+, Rb1^ΔG/ΔG, and Rb1^−/− cells were pulse-labeled with BrdU and harvested at the indicated time points. All graphs represent at least 3 individual experiments, and error bars indicate 1 standard deviation from the mean. An asterisk represents a statistically significant difference from the wild-type control (t test, P < 0.05).