FIG. 4.

The difference in vinblastine-mediated cytotoxicity between wild-type and mekk1^−/− DT40 cells is due to a defect in vinblastine-mediated apoptosis. (A) In DNA laddering assays, 7.5 × 10⁶ DT40 or MC2 cells were treated for 2, 6, or 8 h with 1 μM vinblastine sulfate, for 3.5 h with 12.5 μM etoposide, or for 8 h with DT40 medium alone. DNAs from these samples were prepared as described in Materials and Methods. The equivalent of 3.75 × 10⁶ cells of each sample was run on a 1% agarose gel. (B) In the caspase 3 activity assay, 2 × 10⁷ cells were treated for 2 or 8 h with 1 μM vinblastine sulfate or for 8 h with DT40 medium alone (0 h) and then lysed in 50 μl of ice-cold cell lysis buffer. Extracts were incubated at 37°C with the caspase 3 substrate Ac-DEVD-ρNA. A₄₀₅ readings were taken on a plate reader at 10-min intervals. A₄₀₅ readings (relative caspase activity) at 120 min are shown.