Combination treatment of AC220 and spautins induces metabolic catastrophe. (A) The cellular ATP levels (fold) in scramble (SCR), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 16 h (C43). WB shows the Hsc70 and Lamp2A siRNA knockdown efficiencies. (B) The intracellular ATP content and ADP/ATP ratio of ES2 cells treated with AC220 and/or C43 for 12 h. (C) Relative change in the NAD⁺/NADH ratio in lysates from ES2 cells treated with AC220 and/or C43 for 12 h. (D) The rate of mitochondrial respiration measured by OCR in ES2 cells treated with AC220 and/or C43 for 8 h. (E) Relative change in glutamine levels in the culture medium of ES2 cells treated with AC220 and/or C43 (normalized to cell numbers) for 16 h. (F) The cellular ATP levels in ES2 treated with AC220 and/or C43 in the absence or presence of Oligomycin, Rotenone, Etomoxir, or T0070907 for 16 h. (G) ATP-coupled OCR in scramble (SCR), Hsc70, or Lamp2A siRNA–transfected ES2 treated with AC220 and/or C43 for 8 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, 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.