Fig. 6. Cellular effects of the Bst mutation. (A) Protein synthesis ([³H]leu incorporation) is decreased in Bst/+ MEFs (P<0.001, two-tailed unpaired t-test). (B) Growth rates of subconfluent +/+ and Bst/+ MEF cultures (cell counts), showing decreased doubling time of Bst/+ cells (P<0.001, two-tailed unpaired t-test). (C) Bivariate cell cycle analysis. Cells were sorted by total DNA content (7-AAD fluorescence) and BrdU incorporation (FITC-conjugated antibody). Density plots represent combined data from two experiments (20,000 cells each). The histogram shows the proportion of cells in G1, S and G2/M sectors (red boxes); bars indicate the range for two experiments. The distribution of cells in G1, S and G2/M differ significantly between Bst/+ and +/+ (P<0.001, ²=233 for 2x3 contingency table). (D) S-phase entry of Bst/+ MEFs is delayed following 48 hour serum starvation. [³H]thymidine incorporation was normalized for each genotype to the highest level measured during the experiment. The panel shows one of three independent experiments performed. Each point shows the average of three cultures; bars indicate standard deviation. (E) Model for increased cell cycle length, based on bivariate FACS analysis and doubling times. Assuming the length of S phase is constant, Bst/+ MEFs remain significantly longer in G1 before reaching the restriction point (R).