Figure 2.
Conditional inactivation of Hus1 results in impaired cell proliferation and increased apoptosis. (A) Analysis of cell proliferation. Hus1flox/+ and Hus1flox/Δ1 MEFs at passage one were infectectd with Ad-cre or mock infected and then cultivated following a 3T3 culture schedule as described in Materials and Methods. Plot shows the number of accumulated PDLs. (B) Schematic representation of a FACS dot plot of cell cycle distribution. Staining intensity for PI (x-axis) is plotted versus that for anti-BrdU-FITC (y-axis). The S-phase population is divided into S1 (BrdU positive) and S2 (BrdU negative). (C) Effects of Hus1 loss on cell cycle distribution. MEFs of the indicated genotypes were labeled with BrdU at the indicated times postinfection or mock infection, stained with anti-BrdU and PI, and analyzed by flow cytometry. Cells were passaged 1 d before BrdU labeling. The percentage of cells in each phase of the cell cycle is indicated. (D and E) Increased apoptosis after conditional inactivation of Hus1. MEFs of the indicated genotypes were stained with Annexin V-FITC and PI at the indicated times postinfection or mock infection and analyzed by flow cytometry. (D) Plot shows the percentage of apoptotic cells (Annexin V positive, PI negative). Values are the mean of three independent experiments, with error bars representing the SD. (E) Representative FACS dot plots showing apoptosis in cells of the indicated genotypes at 7 d postinfection or mock infection. Staining intensity for PI (x-axis) is plotted versus that for Annexin V-FITC (y-axis). The percentage of cells categorized as apoptotic (top left quadrant; Annexin V positive, PI negative) or necrotic (top right quadrant; Annexin V positive, PI positive) is indicated.