As₂O₃–dependent degradation of BCR-ABL and induction of autophagy in the BCR-ABL expressing leukemia cells. (A) K562 cells were incubated with As₂O₃ for 24 hours, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (B) K562 cells were incubated with A_S2O₃ at the indicated final concentrations for 24 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. (C) K562 cells were incubated with A_S2O₃ (2μM) in the presence or absence of E64d (10μM), and pepstatin A (10μM) for 24 hours, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. (D) K562 cells were incubated for the indicated times with A_S2O₃ (2μM). Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-p62/SQSMT1 or anti-GAPDH antibodies, as indicated. (E) Electron microscopy analysis for autophagic compartments in untreated K562 cells or cells treated for 24 hours with As₂O₃ (2μM). Data from 5 independent measurements were quantitate and represent autophagic compartment (AC) accumulation in a 100μM² cell surface area. Paired t test analysis showed P = .0207. (F) K562 cells were treated for 24 hours with As₂O₃ (2μM). The cells were then stained with acridine orange for quantitation of formation of acidic vesicular organelles (AVOs). An increase in AVOs formation is accompanied with an increase in FL3/PE-Cy5 fluorescence, reflecting induction of autophagy. Data from 3 independent experiments, including the one shown in the top panel, were quantitated are expressed as means ± SE. Paired t test analysis comparing AS₂O₃–treated cells versus control-untreated cells, demonstrated P = .016.