Figure 2.

Apoptotic features of MBCD-induced cell death. A: A431 cells were treated with 5 mmol/L MBCD, stained with annexin V-FITC and PI, and subjected to flow cytometry analysis. Fluorescence dot blots of annexin V-positive (horizontal axis) and PI-positive (vertical axis) cells are shown. Cells that were positively stained by annexin V-FITC only (early apoptosis) and positive for both annexin V-FITC and PI (late apoptosis) were quantitated, and both subpopulations were considered as overall apoptotic cells. B: A431 cells were treated with 2, 5, and 10 mmol/L MBCD for 4 hours, and DNA was isolated and analyzed by 2% agarose gel electrophoresis. C: Cells were treated with 5 mmol/L MBCD for 4 hours, stained with DAPI, and analyzed by fluorescence microscopy. D: A431 cells were treated without or with 5 mmol/L MBCD for 4 and 6 hours and then incubated with DiOC₆ to monitor ΔΨ_m by FACS. The loss of mitochondrial membrane potential was equated with decreased fluorescence and shift to the left. These experiments were repeated three separate times with comparable results.