FIGURE 2.

Cadein1 efficiently induces apoptosis and G₂/M delay in p53-defective HeLa cells. A, the viability of HeLa, WI-38, and IMR90 cells was evaluated for a range of different concentrations of cadein1 for 24 h by MTT assay. Three independent experiments were performed for each point, and the mean value is plotted with S.D. B, HeLa and WI-38 cells were treated with 6 μm cadein1 for up to 12 h. Cells were analyzed by Western blot with antibodies against cleaved-PARP. C, HeLa and IMR90 cells were treated with 6 μm cadein1 for up to 12 h. Cells were collected every 2 h and analyzed by Western blot with antibodies against PARP and cleaved caspase-3. Actin blots are shown as loading controls. D, DNA contents of HeLa and IMR90 cells were analyzed by flow cytometry, as described under “Experimental Procedures,” and the number of cells in the sub-G₁ phase over the total cells was calculated. The number of cells in sub-G₁ phase (M1) over the total number of cells is plotted as percentages. E, 12 h after release from a double thymidine block, HeLa cells were incubated in the media containing either 4 μm cadein1 or no cadein1 for up to 24 h. DNA contents in HeLa cells were analyzed by flow cytometry at each time point. Cyclin A, cyclin B, and cyclin E are markers of cell cycle progression. Levels of each cyclin at each time point were measured by immunoblots. α-Tubulin was used as a loading control.