Effects of energy stress on ATP depletion, AMPK phosphorylation and apoptosis in CT-2A and astrocyte cell lines. a) Influence of glucose and/or glutamine deprivation on ATP levels in CT-2A astrocytoma and mouse astrocytes. Cells were grown in medium with glucose and glutamine. At 0 hour the medium was replaced with fresh medium without glucose and glutamine and ATP levels were measured at several time points between 0 and 20 hours. ATP levels are presented as relative level normalized to ATP levels in control cells that were given fresh medium with glucose and glutamine at 0 hour. b) Influence of glucose and/or glutamine deprivation on AMPK phosphorylation and caspase-3 cleavage in CT-2A and astrocytes. Cells were treated with different media as indicated for 24 hours. Cell lysates were probed with specific phosphorylated and non-phosphorylated antibodies for western blot as indicated. Increased phosphorylation of AMPK in CT-2A cell was evident with caspase-3 and PARP cleavage in complete absence of serum, glucose, and glutamine. c) Influence of glucose and/or glutamine deprivation on Annexin positive apoptotic cells. Cells were incubated in the presence and absence of glucose and glutamine containing media for 18 hours. Cells positive either for Annexin V or for both Annexin V and PI were considered early apoptotic and late apoptotic cells, respectively. Glucose/glutamine withdrawal induced apoptosis in 48–50% of CT-2A cells but not in astrocytes. d) Influence of glucose and/or glutamine deprivation on caspase 9 cleavage in CT-2A cells. Cells were treated with different media as indicated and incubated for 0–8 hours. Cell lysates were probed with anti-caspase 9 for western blot. Cleavage of caspase-9 started at 6 hours in complete absence of glucose and/or glutamine. e) Caspase-9 cleavage was associated with cytochrome c release at 8 hours in the cytosolic fraction of CT-2A cells. f) Influence of 3.0 mM glucose on AMPK phosphorylation in CT-2A and astrocytes. Cells were treated for different hours as indicated. AMPK was maximum phosphorylated in CT-2A cells at 18 hours and started dying in 24 hours. AMPK phosphorylation was greater in CT-2A than in astrocytes. g) Influence of 2-deoxyglucose (2-DG) on AMPK phosphorylation in CT-2A and astrocytes. Cells were treated with 50 mM conc. of 2-DG from 0–30 minutes in serum free media. Western blot analysis for phosphorylated AMPK revealed increased phosphorylation in CT-2A cells compared to astrocytes. h) Increase in AMPK phosphorylation with time (analyzed from g). All experiments were done in triplicate.