FIG. 4.
Kinetics of Bid cleavage, reduction in ΔΨm, and cytochrome c release in Stx1-treated THP-1 cells. Cells were harvested at 0, 1, 2, 4, 6, and 8 h after Stx1 treatment. (A) Cell lysates were prepared, and the uncleaved (22-kDa) form of Bid was detected in the lysates by immunoblotting (upper panel). Levels of Bid were quantitated by densitometric scanning and compared to control levels (lower panel). Data shown are the means ± standard errors of the means from two independent experiments. *, a significant difference (P < 0.02) compared to control cells. (B) Cells were collected after Stx1 treatment, and the JC-1 reagent was used to measure reductions in ΔΨm by flow cytometry. Percentages of cells staining for red and green fluorescence (upper right quadrants) and red fluorescence only (lower right quadrants) are shown in the upper right-hand corners of the quadrants. A decrease in ΔΨm is associated with a shift in reactive cells from the upper right quadrant to the lower right quadrant. Representative scatter plots from two independent experiments are shown. (C) Cytosolic fractions were prepared, and cytochrome c was detected in the fractions by immunoblotting. A representative blot from two independent experiments is shown.