AML cells have high levels of constitutive autophagy, compared with normal CD34⁺cells and are sensitive to autophagy inhibition. Immunoblot analysis of the indicated AML cell lines (A) and patient samples (B) of LC3 and actin (loading control) showed different levels of basal autophagy. Quantification of LC3-I and -II levels was performed with Image J software. (C) The KG-1 and HL60 cell lines were incubated with NH₄Cl overnight (>12 hours), and the lysates were analyzed for LC3 and actin (loading control). Increased LC3-II after treatment indicates active autophagic flux. (D) CD34⁺ cells from CB, BM, or patients with AML were stained with antibodies against LC3 (green) as a marker of autophagosomes or LAMP-2 (red) as a marker of lysosomes with DAPI (blue) as a nuclear stain. (E) Electron micrograph images show more autophagosomes in AML cells, compared with nonmalignant CB cells. Original magnification ×15 000, ×30 000 zoom. (F) Samples from AML patients were treated with increasing concentrations of autophagy inhibitors CQ, 3-MA, or Baf A1. Cell viability was measured with YOPRO-1 and 7-AAD and is displayed as the percentage of viable cells, compared with the untreated controls.