Clonogenic growth after paclitaxel withdrawal is enhanced in MEFs lacking caspase-9 or Apaf-1 expression. (A) Clonogenicity assay of wild-type, caspase-9^−/− and Apaf-1^−/− MEFs. Cells were treated with the indicated concentrations of paclitaxel. After 48 hours, cells were washed, split 1:10, and kept in normal medium. After 7 days of further incubation, cells were stained with crystal violet and subsequently scanned using an Epson Perfection 3200 scanner and processed using Adobe Photoshop 7.0 software (Adobe, San Jose, CA) (right panel). The relative proliferation compared with untreated controls was calculated as the mean (± SD) by measurement of the dye absorbance (left panel). (B) Caspase-9– and Apaf-1–deficient cells, but not wild-type cells, retain their proliferative capacity. Cells were treated as described in panel A and analyzed by light microscopy using a Zeiss Axiovert 135 microscope (40×/0.6 Korr-objective), a ProgResC14 digital camera, and Openlab 3.5.1 software. (C) After prolonged cultivation of paclitaxel-treated caspase-9– or Apaf-1–deficient cells, a proportion of the cells undergo senescence as assessed by staining for senescence-associated β-galactosidase (blue). Staining of treated (1 μM paclitaxel for 48 hours) and untreated cells was analyzed after further cultivation in normal medium for 2 weeks. (D) Even long-term paclitaxel treatment triggers mitochondrial depolarization (right panel) and cell death (left panel) only in wild-type cells, but not in caspase-9– or Apaf-1–deficient cells. In wild-type cells, these effects are partially caspase dependent. MEFs were incubated with 1 μM paclitaxel for 4 days and subsequently analyzed by flow cytometry. Results show the mean values (± SD).