Hypoxia reduces chemosensitivity in AML cell lines, which can be restored with autophagy inhibition. The indicated AML cell lines were treated with Ara-C (100 nM HEL-Luc, 250 nM HL60, and MOLM-13 in normoxia (21% O₂) or hypoxia (1% O₂). Concentrations were determined based on cell line sensitivity to Ara-C. (A) Cells were processed after 72 hours for measurement of apoptosis by flow cytometry with annexin V/7-AAD or PI staining. Data are shown as a percentage of annexin V– and/or 7-AAD– or PI-positive cells normalized to untreated cells (≥3 biological replicates; 2 technical replicates). (B) HEL-Luc cell lines were treated with 100 nM Ara-C for 72 hours, and the total number of cells was quantified (4 biological replicates). (C) HEL-Luc cell lines were treated with 100 nM Ara-C, and phospho-H2A.X flow cytometry was performed after 48 hours. Data are shown as the MFI of p-H2A.X staining normalized to untreated in normoxia (2 biological replicates). (D-E) MOLM-13 and HL60 cells were treated with 250 nM Ara-C in normoxia (21% O₂) or hypoxia (1% O₂) in combination with bafilomycin A1 (Baf A1; 2 nM) (D) or chloroquine (CQ; 25 µM) (E). The cells were processed after 72 hours for apoptosis flow cytometry, using annexin V/7-AAD or PI staining. Data are shown as a percentage of annexin V– and/or 7-AAD– or PI-positive cells (3 biological replicates; 2 technical replicates). Error bars represent ± standard deviation. *P < .05; **P < .01; ***P < .001.