FIG. 9.
Cell death following glucose limitation mimics cell death induced by growth factor withdrawal. (A) Control (Neo) and Bcl-xL-expressing cells cultured with 0.35 ng of IL-3/ml were washed and resuspended in medium containing the same amount of IL-3 but only 0.02 mM glucose. The mean cell viability (and standard error of the mean [SEM]) following glucose limitation over time is shown. (B) Mitochondrial potential was assessed using the potentiometric dye TMRE with cells growing for 6 h in the presence or absence of IL-3 (upper panel) or with high or low concentrations of glucose (10 or 0.02 mM, respectively) (lower panel). Note that the same histogram is used for the 10 mM glucose plus IL-3 condition in both panels. (C) Bax conformation was detected in mitochondrial fractions from cells growing for 15 h in the presence (+IL-3) or absence (−IL-3) of IL-3 or with 0.02 mM glucose. Mitochondrial lysates were prepared, immunoprecipitated (IP) using a conformation-specific anti-Bax antibody, and immunoblotted (IB) for Bax. (D) Control (Neo) and Bcl-xL-expressing cells were cultured with 0.35 ng of IL-3/ml in the presence of 11 mM glucose (Control) or 0.02 mM glucose (glucose withdrawal) for 18 h prior to subcellular fractionation. The amounts of cytochrome c (Cyt. c) and cytochrome oxidase subunit IV (Cox IV) present in the mitochondrial (M) and cytosolic (S) fractions were determined by Western blotting. Cox IV, an integral membrane protein located in the inner mitochondrial membrane, served as a control to demonstrate the absence of mitochondria in the cytosolic fraction.