Paclitaxel-induced apoptosis depends on caspase-9 and Apaf-1. (A) Left panel shows status of caspase-9 or Apaf-1 expression in wild-type and the caspase-9^−/− and Apaf-1^−/− MEFs as assessed by immunoblot analysis. (B) Caspase-9 and Apaf-1 knock-out MEFs are resistant to paclitaxel-induced apoptosis. Wild-type, caspase-9^−/−, or Apaf-1^−/− MEFs were treated with increasing concentrations of paclitaxel for 48 hours or left untreated (left panel). Apoptosis was assessed by measurement of hypodiploid nuclei and is given as the mean (± SD). Right panel shows that apoptosis in wild-type MEFs treated with paclitaxel is blocked by the caspase inhibitor QVD-oPh (20 μM). (C) Paclitaxel treatment disrupts the mitochondrial membrane potential (left panel) and triggers PS exposure (right panel) in wild-type, but not in caspase-9– or Apaf-1–deficient cells. These effects are largely caspase dependent. Cells were treated with 1 μM paclitaxel for 48 hours in the presence or absence of QVD-oPh. Results show the mean values (± SD). (D) Paclitaxel induces the formation of multipolar spindles with lagging and misaligned chromosomes independently of caspase-9 or Apaf-1 expression. Microtubuli (green) were detected by immunofluorescence staining with anti–α-tubulin and chromosomes (blue) with the DNA dye DAPI and analyzed by confocal microscopy. (E) Active caspase-3 is only detected in paclitaxel-treated wild-type, but not in caspase-9– or Apaf-1–deficient MEFs. Exposure of the caspase-3 blot was increased from 30 seconds to 2 minutes in order to detect active caspase-3 fragments (◁). Noncleaved caspase-3 and actin are marked (◀). (F) DEVDase activity is not induced in paclitaxel-treated MEFs deficient in caspase-9 or Apaf-1. Results are shown as mean values (± SD).