Treatment with AC220 (Quizartinib) reduces glycolysis and induces macroautophagy. (A) Proliferation capacity (%) of ES2 and Sum159 cells treated with AC220 for 16 h. Phospho- and total Akt levels of ES2 and Sum159 cells treated with AC220 up to 24 h. (B) WB of phospho- and total Akt levels of ES2 cells treated with AC220 and C43 (left), or C43 alone in confluent (Conf) or nonconfluent (Non-C) conditions (right) for 24 h. (C) Relative change in glucose levels in the culture medium of ES2 cells treated with AC220 and/or C43 (normalized to cell numbers) for 16 h. (D and E) The glycolytic activity and maximum glycolytic capacity of ES2 (D) or Molm-14 (E) cells, determined by ECAR, after AC220 and C43 treatment for 12 h (ES2) or 8 h (Molm-14). (F) Glucose flux analysis using [U¹³C]glucose. A schematic depiction of intermediary metabolites of glycolysis is shown. ¹³C enrichment of intracellular glucose-derived metabolites, marked in bold, is presented. (G) WB of LC3 protein levels in ES2 cells, treated with increasing concentrations of AC220 and/or 5 µM E64D for 16 h (top), or treated with C43 or A70 in combination with AC220 and/or 5 µM E64D for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle), 1 µM AC220, 10 µM C43, or 1 µM A70, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise shown. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.