Fig. 5

Complete inhibition of glucose uptake promotes Complex I dependency for survival. A NB4, THP-1, and MOLT-4 were treated with DMSO or KL-11743 (500 nM) for 24 h. Heavy membranes were isolated, digitonin-extracted, and the protein complexes were resolved using native PAGE conditions. In-gel activity was stimulated with NADH and the arrow above 720 kDa indicates assembled CI; the right panel is a Coomassie Blue loading control. B Quantification of the main bands indicated by the arrow (ASSEMBLED) and the activity detected below the assembled bands (FREE) in A. Numbers depicted in bars show the % of total activity attributed to the assembled CI species. C NB4, THP-1, and MOLT-4 were treated with DMSO or KL-11743 (500 nM) for 24 h, and total RNA was harvested. The fold change of transcripts from mitochondria encoded genes were measured by real-time qPCR. Expression was normalized against 18S. D CI analysis of NB4, THP-1, and MOLT-4 treated with DMSO or IACS (10 nM) for 24 h. OCR was measured by an Agilent XFe96 Analyzer during sequential administration of a PMA: pyruvate (10 mM) + malate (0.5 mM) + ADP (4 mM), R: rotenone (1 μM), S: succinic acid (10 mM), and F: FCCP (1 μM). E NB4, THP-1, and MOLT-4 were treated with DMSO, KL-11743 (500 nM), or IACS (1, 3, 6, 10 nM) ± KL-11743 (500 nM) for 24 h. Apoptosis was measured by AV labeling and flow cytometry. CHX (50 μg/mL) + ABT-737 (1 μM) is a positive control for apoptosis. F NB4, THP-1, and MOLT-4 were treated as with DMSO, KL-11743 (500 nM), or IACS (1, 3, 6, 10 nM) ± KL-11743 (500 nM), imaged every 2 h, and analyzed for YOYO3 + cells; the mean YOYO3 + events per image of 2 replicates is presented. G NB4, THP-1, and MOLT4 were treated with DMSO or GSK-1120212 (25 nM) for 24 h before PI staining and flow cytometry. (H) NB4, THP-1, and MOLT4 were treated with GSK-1120212 (25 nM), KL-11743 (500 nM), or IACS (10 nM) for 24 h, and ECAR (basal versus glucose-stimulated, 10 mM) was measured. (I) NB4, THP-1, and MOLT4 cells were treated with GSK-1120212 (25 nM) ± IACS (10 nM) for 24 h. Apoptosis was measured by AV labeling and flow cytometry. Error bars are the average of 3–6 technical replicates ± SEM