Hypoxia induces autophagic flux in AML cell lines. AML cell lines were incubated in normoxia or hypoxia for 48 hours, with or without lysosomal protease inhibitors (10 µg/mL pepstatin A and E-64d) added for the final 6 hours. (A) Immunoblot analysis of LC3 and actin (loading control) is shown. LC3-II levels were determined by densitometric analysis and normalized to actin. Values are relative to untreated cells in 21% O2. (B) Immunoblot analysis of p62 and actin (loading control). Blots are representative of 2 independent experiments. (C) HEL-Luc cells were stained with Cyto-ID, fixed, mounted in Vectashield+DAPI, and imaged on a Leica TCS SP2 spectral confocal microscope with a 63× objective (blue, nucleus/DAPI; green, autophagosomes/Cyto-ID). Bar represents 10 µm. (D) Microscopic images were quantified by ImageJ to determine the percentage of Cyto-ID+ cells (3 biological replicates, ≥100 cells counted per replicate). Error bars represent ± standard deviation. *P < .05.