MTA-induced apoptosis involved mitochondria (A) MTA induced mitochondrial membrane damage in human melanoma cells as indicated by ∆Ψm collapse. Cells were incubated with 0.84 μM MTA for the indicated times. Subsequently, the mitochondrial membrane potential was measured by staining the cellls with 40 nM DiOC6. In the histogram, the curve shifted to the right, which indicates retention of the dye in the mitochondria (an early feature of apoptosis), followed by shifting of the curve to the left after 48 h due to collapse of the mitochondrial membrane potential. This figure is representative of 3 independent experiments. Similar values were obtained for the 4 melanoma cell lines and Calu-3 (data not shown). (B) MTA induced the release of cyt c and AIF from mitochondria. Cells were treated with 0.84 μM MTA for 48 h before immunocytochemistry. In untreated cells, cyt-c and AIF (green) exhibited a punctuate pattern which corresponds to the mitochondrial localization of these proteins. After 48 h of MTA exposure cyt-c and AIF immunoreactivity consisted of a diffuse pattern, indicative of their localization in the cytosol after the released from the mitochondria. This diffuse pattern of immunoreactivity was seen in cells which presented a fragmented nucleus (blue), indicative of apoptosis. (C) Immunoblotting of cell lysates from cytosolic and mitochondrial extracts of cells treated with 0.84 μM MTA for the indicated time periods using antibodies specific for the indicated proteins. The blots were stripped and reprobed with actin which served as loading control. Cyt c and AIF levels can be seen to decrease in the mitochondrial fraction while concomitantly increasing in the cytosolic fractions of MTA-treated cells.