Figure 4.

Translocation of cytochrome-c from the mitochondrial to the cytosolic compartment in butyrate treated Caco-2 cells. (A) After ultracentrifugation to separate the mitochondrial from the cytoplasmic compartments, a shift of cytochrome-c was observed by western blot in response to butyrate. In contrast, a clear and strong signal for apoptosis inducing factor (AIF) in western blots was only observed in the mitochondrial compartment, without any shift of AIF to the cytosolic compartment, even at high butyrate concentrations. (B) In addition, immunofluorescence analysis confirmed that the perinuclear mitochondrial staining for cytochrome-c in unstimulated cells changed to a diffuse pattern of cytosolic staining in cells after stimulation with butyrate. (C) Inhibition of the caspase cascade with the peptide inhibitor zVAD-fmk (200 μM) did not alter the translocation of cytochrome-c, indicating that this event was independent and upstream of the caspase cascade. One of three representative experiments is shown for each blot.