(A) Methyl pyruvate suppresses the thalidezine-mediated autophagic effect in cancer cells. HeLa cells were treated with 10 μM of thalidezine (Tha) with or without 5 mM of methyl pyruvate (MP) for 24 h. Representative micrographs of autophagic cells and cell percentage with EGFP-LC3 puncta formation quantification. Scale bar = 10 μm, 60X. (B) Methyl pyruvate abolishes the thalidezine-mediated LC3-II conversion. Immunoblot for LC3-I, LC3-II, and β-actin detection (Uncropped blots images, Supplementary Figure 4B). (C) Methyl pyruvate abrogates thalidezine-mediated cell death. Annexin V stain flow cytometry analysis (lower panel) and percentage of cell death quantification (upper panel). (D) Thalidezine inhibits mitochondrial respiration and oxygen consumption rate (OCR). (E) Thalidezine inhibits glycolysis and extracellular acidification rate (ECAR). HeLa cells were treated with 15 μM of thalidezine for 24 h and then subjected to seahorse analysis using OCR and ECAR assay kits. (F & G) Thalidezine decreases the ATP production in HeLa and DLD-1 BAX-BAK DKO colon cancer cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Data were mean value ± S.D of three independent experiments.