FIGURE 7.

GM-DCs activated in the presence of mTOR inhibitors are insensitive to glucose deprivation but are sensitive to disruption of fatty acid catabolism. (A) GM-DCs were either left unstimulated or activated with LPS for 18 h in the presence or absence of RAP or KU. Cultures were washed and then media replaced with complete media, glucose-free media, or glucose-free media supplemented with galactose. The percentage inhibition of ATP levels compared with the complete media group 30 min after media change is depicted. (B) DCs were treated as in (A) and cell viability was monitored 24 h after media change. (C and D) Cells were treated as indicated for 24 h and then plated overnight at 5 × 10⁴ cells per well onto PMM Tox-1 plates containing either glucose (C) or pyruvic acid (D) as the primary carbon substrate in nutrient-restricted media (no glucose, 5% FCS, 0.3 mM l-glutamine). NADH-reactive redox dye was added 18 h later and dye reduction by NADH was measured at 540 nm absorbance at the indicated times. Data are normalized to absorbance readings at time 0. (E) DCs were either untreated or stimulated with LPS in the presence or absence of KU. After 24 h, culture media was changed to glucose-replete media, glucose-free media containing galactose, or glucose-free media containing galactose in the presence of a fatty acid transporter inhibitor (etomoxir [eto]) or a glutamine antagonist (6-diazo-5-oxo-l-norleucine [DON]). Cellular ATP levels were monitored 4 d after media change. Graphs in this figure represent mean values ± SD of technical replicates that are representative of at least two independent experiments. *p < 0.05.