Figure 1.

Loss of Chk1 and PTEN abrogates recovery from stalled DNA replication. A, schematic presentation of the experiments. Human osteosarcoma U2OS cells were transfected with scrambled siRNA (control, 10 µmol/L) or siRNA specific for Chk1 (20 µmol/L) and PTEN (20 µmol/L). Next day, HU was added to the cell culture medium (100 µmol/L). After 18 h, cells were washed with PBS three times, and fresh medium was supplied. Cell cycle profiles were analyzed every 24 h for the next 3 d. B, U2OS cells transfected with scrambled siRNA were used as controls for fluorescence-activated cell sorting analysis to determine the alignment of G₁, S, and G₂-M phases. The fraction of cells in each cell cycle is shown for each sample. Profiles of +HU are the results of fluorescence-activated cell sorting analysis after treatment with HU for 18 h. C, immunoblot analysis of Chk1 and PTEN for each time point. Tubulin was used as a loading control. D, MCF7 cells were transfected with control or Chk1-siRNA as described above and treated with HU similarly. Cells were then washed with PBS and fluorescence-activated cell sorting analysis was done after 72 h. Each cell cycle population was determined as U2OS cells. Levels of Chk1, PTEN, and phospho-PTEN (P-PTEN) are also indicated.