Figure 1.
Mitochondrial and nuclear features of apoptosis in Apaf-1−/− and caspase-3−/− cells. MEFs from control mice (wild-type), Apaf-1−/−, or caspase-3−/− knockout mice were treated for 24 h with 2 μM STS (A and B), 100 μM etoposide, 150 μM cisplatin, 50 μM arsenite (B) and/or 50 μM Z-VAD.fmk (right panels only), followed by determination of the fall of the ΔΨm (measured as a reduction of the red fluorescence emitted by the ΔΨm-sensitive dye JC-1), mitochondrio-nuclear, and mitochondrio-cytosolic translocation of AIF or Cyt-c respectively (determined by immunostaining), proteolytic activation of caspase-3 (detected with an antibody specific for active caspase-3), or nuclear morphology (detected with Sytox-green). Individual cells demonstrated in A are shown after 8 h (wild-type) or 24 h (Apaf-1−/−, caspase-3−/−) of treatment with STS and are representative for the dominant phenotype. The chromatin condensation of wild-type cells cultured with STS was regarded as stage II of nuclear apoptosis, whereas Apaf-1−/− or caspase-3−/− cells manifest a stage I pattern of chromatin condensation. Kinetic analyses of apoptotic parameters induced by STS (▵), etoposide (□), cisplatin (▪), arsenite (○) and/or Z-VAD.fmk (right panels) are shown in B. This experiment has been repeated three times, yielding comparable results. Electron microscopy (C), large scale DNA fragmentation (D), and oligonucleosomal DNA fragmentation (E) of cells treated as in A are also shown.
