Fig. 2: Mitochondrial respiration, but not glycolysis, is responsible for reduced drug accumulation in chemoresistant cells.

a, b NCI/ADR-RES cells were incubated with or without (Veh) oligomycin (Oligo, 5 μM) or rotenone (Rote, 50 μM) for 2 h followed by incubation with doxorubicin (Dox, 3 μM) for 3 h. Cells were then fixed, stained with DAPI (nuclear dye, blue), and analyzed by confocal microscopy for doxorubicin fluorescence (red). a Representative images. Scale bars represent 20 μm. b Quantification of doxorubicin intensity relative to nuclear area (vehicle n = 12, olligomycin n = 12, rotenone n = 17). p = 0.0001, 0.0001. c Cell death analysis using the Live/Death staining and flow cytometry analysis in cells after the treatments described in (a). Numbers represent the percentage of dead cells. d, e NCI/ADR-RES cells were incubated with increasing concentrations of d oligomycin (p = 0.0001, 0.0001) or e rotenone (p = 0.0006, 0.0001) as indicated for 2 h followed by incubation with doxorubicin (3 μM) as in (a). Cells were then fixed and analyzed for doxorubicin fluorescence by flow cytometry. Median fluorescence intensity (MFI) is shown (n = 3). f, g NCI/ADR-RES cells were incubated with oligomycin (5 μM) or 2-deoxyglucose (2-DG, 50 mM) for 2 h or without glucose (No Gluc) for 24 h followed by incubation with doxorubicin (3 μM) as in (a). Cells were then fixed, stained, and analyzed as in (a, b). f Representative images. Scale bars represent 20 μm. g Quantification of doxorubicin intensity relative to nuclear area (vehicle n = 176, 2-DG n = 75, no glucose n = 46, olligomycin n = 109). p = 0.0001, 0.9918, 0.9999. h NCI/ADR-RES cells were incubated with increasing concentrations of 2-deoxyglucose as indicated for 2 h followed by incubation with doxorubicin as (3 μM) in (a). Cells were then fixed and analyzed as in (e, f) (n = 3). p = 0.3372, 0.0012. i MES/Dox cells were incubated with oligomycin (5 μM), rotenone (50 μM), or 2-deoxyglucose (50 mM) for 2 h followed by incubation with doxorubicin as in (a). Cells were then fixed and analyzed as in (d, e) (n = 3). p = 0.0001, 0.0001, 0.967. j NCI/ADR-RES cells were incubated with oligomycin (5 μM) or 2-deoxyglucose (50 mM) for 5 h and then total ATP levels were determined by Luciferase assay (n = 4). ATP concentration (10⁴ cells) is shown. p = 0.0001, 0.0001. k NCI/ADR-RES cells were incubated with 2-DG (50 mM), CB-839 (5 μM) or both for 2 h followed by incubation with doxorubicin (3 μM), and analyzed for doxorubicin fluorescence by flow cytometry. p = 0.479, 0.1162, 0.0003. Mean ± SEM is provided for all figures. * denotes p < 0.05 by one-way ANOVA and Tukey’s multiple comparisons test. Results are representative of at least two experiments.