Sensitivity to autophagy inhibitors in hypoxia correlates with the extent of inhibition of mitochondrial respiration. (A-B) MOLM-13 AML cell lines were treated with the indicated concentrations of CQ (A) or Baf A1 (B) in normoxia or hypoxia for 72 hours and analyzed by annexin V/PI flow cytometry (≥3 biological replicates; 2 technical replicates). (C-D) MOLM-13 cells were transfected with siRNAs against Atg-5 and Atg-7. After 24 hours, they were placed in normoxia or hypoxia. Lysates harvested after 48 hours for immunoblot analysis (C) (quantification under each blot normalized to actin, followed by normalization to NT in normoxia), or the cells were analyzed after 72 hours by annexin V/PI flow cytometry (D) (3 biological replicates; 2 technical replicates). (E-F) MOLM-13 cells were treated with CQ (E) or Baf A1 (F) for 48 hours in normoxia or hypoxia and analyzed on a Seahorse XF analyzer. Representative MitoStress Test profiles and average maximal respiration for cells are shown (2 biological replicates; 3 technical replicates). (G-H) MOLM-13 cells were treated with CQ (G) or Baf A1 (H) for 48 hours, stained with MitoID, and analyzed by flow cytometry (3 biological replicates; 2 technical replicates). Data are shown as average mean fluorescence intensity (MFI). (I) MOLM-13 cells were harvested after a 48-hour treatment with 10 µM CCCP, 50 µM CQ, or 25 nM Baf A1 and blotted for PINK-1. The levels were determined by densitometric analysis and normalized to actin. Values are shown relative to DMSO. (J) MOLM-13 cells were treated with 10 µM CCCP or 50 µM CQ, alone or in combination in normoxia or hypoxia for 72 hours, and analyzed by annexin V/PI flow cytometry (3 biological replicates; 2 technical replicates). Error bars represent ± standard deviation. *P < .05; **P < .01; ***P < .001; ****P < .0001.