Arsenic trioxide-induced autophagy in leukemic cell lines. A, KT1 cells were incubated with As₂O₃ at the indicated concentrations for either 8 or 48 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. UT, untreated cells incubated for 48 h in parallel. B, KT1 cells were treated with arsenic trioxide (2 μm) for the indicated times. Cells were lysed, and total lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. UT, untreated cells incubated for 120 min in parallel. C, KT1 cells were pretreated for 60 min with pepstatin A (10 μm) and/or E64-d (10 μm) as indicated and were subsequently treated for 24 h with As₂O₃ (2 μm) in the continuous presence or absence of the inhibitors, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-LC3 or anti-GAPDH antibodies, as indicated. D, KT1 cells were treated with As₂O₃ (2 μm) for the indicated times. Total lysates were resolved by SDS-PAGE and immunoblotted with anti-p62 or anti-GAPDH antibodies, as indicated. UT, untreated cells incubated for 48 h in parallel. E, U937 cells were treated for 24 h with the indicated concentrations of As₂O₃. Total lysates were resolved by SDS-PAGE and immunoblotted with the antibodies against anti-LC3, anti-p62, or anti-GAPDH, as indicated. F, KT1 cells were either transiently transfected with LC3-GFP (upper and lower right panels) or with control empty vector (upper and lower left panels). Samples were either not treated or treated with As₂O₃ (2 μm) for 24 h. Cells were stained with anti-GFP, and signals were detected by confocal microscopy. Punctated LC3 seen at lower right panel is a characteristic of autophagosome formation. UT, untreated.

As₂O₃-dependent formation of AVOs in leukemic cells. Upper panel, KT1 cells were pretreated for 60 min with UO126 (10 μm) or SP600125 (10 μm) and were subsequently treated for 24 h with As₂O₃ (2 μm). Cells were then stained with acridine orange for quantitation of formation of AVOs. An increase in AVO formation is accompanied with an increase in FL3/PE-Cy5 fluorescence, reflecting induction of autophagy. Lower panel, data in the lower panel are from quantitation of four independent experiments, including the one shown in the upper panel, and are expressed as means ± S.E. Paired t test analysis comparing As₂O₃-treated cells versus untreated (UT) cells or versus As₂O₃ + UO126-treated cells demonstrated paired values of p = 0.0128 and p = 0.0251, respectively.

Induction of autophagy is essential for generation of the antileukemic effects of As₂O₃ on primitive leukemic progenitors from AML patients. A, effects of As₂O₃ (0.5 μm) or chloroquine (CQ) (2.5 μm) or the indicated combinations on primitive leukemic progenitor (CFU-L) colony formation from different AML patients were examined in clonogenic assays in methylcellulose. Data are expressed as percent control (Ctrl) CFU-L colony formation for untreated cells and represent means ± S.E. of three independent experiments. Paired t test analysis comparing the effects of As₂O₃ in the absence or presence of chloroquine showed a paired p value = 0.0261. B, effects of beclin 1-specific siRNA on CFU-L colony formation in the presence or absence of As₂O₃ (0.5 μm). Data are expressed as percent control of CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of three independent experiments performed with samples from different AML patients. Paired t test analysis comparing the effects of As₂O₃ in cells transfected with beclin 1 siRNA versus cells transfected with control siRNA showed a paired value p = 0.0147. C, effects of Atg7-specific siRNA on CFU-L colony formation in the presence or absence of As₂O₃ (0.5 μm). Data are expressed as percent control of CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of four independent experiments. Paired t test analysis comparing the effects of As₂O₃ in cells transfected with Atg7 siRNA versus cells transfected with control siRNA showed a paired value = 0.049.

Engagement of the MEK/ERK pathway is essential for As₂O₃-induced autophagy. A, KT1 cells were treated with As₂O₃ (2 μm) for the indicated times. Total lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of ERK on Thr-202/Tyr-204 or anti-ERK antibodies, as indicated. B, upper panel, KT1 cells were pretreated for 60 min with UO126 (10 μm) or SP600125 (10 μm) and were subsequently treated for 24 h with As₂O₃ (2 μm) or diluent control (DMSO). Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Lower panel, signals for the bands corresponding to LC3-II and GAPDH were quantitated by densitometry, and data were expressed as ratios of LC3-II over GAPDH. Means ± S.E. of three independent experiments are shown, including the one shown in the upper panel. C, KT1 cells were transiently transfected with LC3-GFP. Samples were pretreated for 60 min with UO126 (10 μm) or SP600125 (10 μm) and were subsequently treated for 24 h with As₂O₃ (2 μm) or diluent control (DMSO). Cells were stained with anti-GFP, and punctated LC3 signals were detected by confocal microscopy. D, KT1 cells were transfected with control siRNA or siRNAs specifically targeting MEK1 and/or MEK2 and subsequently treated for 24 h with As₂O₃ (2 μm), as indicated. Cells were lysed, and total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3, anti-MEK1, anti-MEK2, or anti-GAPDH antibodies, as indicated. E, upper panel, Akt1/2^+/+ and Akt 1/2^−/− MEFs were treated with As₂O₃ (2 μm) for 24 h. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. Lower panel, signals for the bands corresponding to LC3-II and GAPDH were quantitated by densitometry, and data were expressed as ratios of LC3-II over GAPDH. Means ± S.E. of three independent experiments are shown, including the one shown in the upper panel. F, KT1 cells were pretreated for 60 min with rapamycin (Rap) (20 nm) and were subsequently treated for 24 h with As₂O₃ (2 μm). Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. G, KT1 cells were pretreated for 60 min with DTT (1 mm) and were subsequently treated for 24 h with As₂O₃ (2 μm). Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. H, KT1 cells were pretreated for 60 min with DTT (1 mm) or N-acetylcysteine (NAC) (10 mm) and were subsequently treated for 24 h with As₂O₃ (2 μm). Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. UT, untreated.

Pharmacological inhibition of autophagy with chloroquine partially reverses the suppressive effects of As₂O₃ on KT1- or U937-derived CFU-L. A, KT1 cells were plated in a methylcellulose assay system in the presence of either As₂O₃ (0.5 μm) and/or chloroquine (CQ) (2.5 μm), as indicated. Data are expressed as percent control (Ctrl) of CFU-L colony numbers for control untreated cells and represent means ± S.E. of four independent experiments. Paired t test analysis comparing the effects As₂O₃ in the absence or presence of chloroquine showed a paired p value = 0.0052. B, KT1 or U937 cells were transfected with control siRNA or siRNAs specifically targeting beclin 1 or Atg7, as indicated. Cells were lysed, and total lysates were resolved by SDS-PAGE and immunoblotted with anti-beclin 1, anti-Atg7, or anti-GAPDH antibodies, as indicated. C, KT1 (left panel) or U937 (right panel) cells were transfected with control siRNA or siRNAs specifically targeting Atg7, as indicated. Cells were then treated for 24 h with As₂O₃ (2 μm), and total lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-GAPDH antibodies, as indicated. D, U937 cells were transfected with control siRNA or beclin 1 siRNA or Atg7 siRNA as indicated, and the effects of As₂O₃ (0.5 μm) on CFU-L colony formation were assessed in clonogenic assays in methylcellulose. Data are expressed as percent control of CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of four independent experiments. Paired t test analysis comparing As₂O₃-treated control siRNA-transfected cells versus As₂O₃-treated beclin 1 siRNA-transfected cells or versus As₂O₃-treated Atg7 siRNA-transfected cells demonstrated paired values of p = 0.005 and p = 0.0109, respectively. E, U937 cells were transfected with control siRNA or beclin 1 siRNA-2 as indicated, and the effects of As₂O₃ (0.5 μm) on CFU-L colony formation were assessed in clonogenic assays in methylcellulose. Data are expressed as percent control of CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of three independent experiments. Paired t test analysis comparing As₂O₃-treated control siRNA-transfected cells versus As₂O₃-treated beclin 1 siRNA-2-transfected cells demonstrated a paired value of p = 0.0373. F, KT1 cells were transfected with control siRNA or Atg7-siRNA as indicated, and the effects of As₂O₃ (0.5 μm) on CFU-L colony formation were assessed in clonogenic assays in methylcellulose. Data are expressed as percent control of CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of four independent experiments. Paired t test analysis comparing As₂O₃-treated control siRNA-transfected cells versus As₂O₃-treated Atg7 siRNA-transfected cells demonstrated a paired value of p = 0.0498. G, KT1 cells were transfected with control siRNA or beclin1-siRNA as indicated, and the effects of As₂O₃ (0.5 μm) on CFU-L colony formation were assessed in clonogenic assays in methylcellulose. Data are expressed as percent control CFU-L colony numbers for control siRNA-treated cells and represent means ± S.E. of four independent experiments. Paired t test analysis comparing the effects of As₂O₃-treated control siRNA transfected cells versus As₂O₃-treated beclin 1 siRNA-transfected cells demonstrated a paired value of p = 0.0048. H, KT1 cells were transfected with control siRNA or beclin 1 siRNA-2 as indicated, and the effects of As₂O₃ (0.5 μm) on CFU-L colony formation were assessed in clonogenic assays in methylcellulose. Data represent means ± S.E. of three independent experiments. Paired t test analysis comparing the effects of As₂O₃-treated control siRNA-transfected cells versus As₂O₃-treated beclin 1 siRNA-2-transfected cells demonstrated a paired value of p = 0.0403.