FIGURE 1 from Autophagy Inhibition–induced Cytosolic DNA Sensing Combined with Differentiation Therapy Induces Irreversible Myeloid Differentiation in Leukemia Cells

Combined treatment with MRT and ATRA induces the irreversible differentiation of human leukemia cells by activating the AIM2 inflammasome. A, HL-60 cells were treated with the indicated concentrations of MRT in the presence or absence of 1 µmol/L ATRA for 48 hours. Fold change in cell number was calculated by dividing the values of the treated samples by the mean value of the untreated control. Data represent the mean ± SD from four independent experiments. B, Giemsa staining of HL-60 cells 48 hours after combined or single treatment with 1 µmol/L ATRA and 1 µmol/L MRT. Representative results from three independent experiments are shown here. C, Expression of CD38 and CD11b on untreated (black dots) and treated [with ATRA, MRT, or ATRA+MRT for 24 hours (blue dots) or 48 hours (red dots)] HL-60 cells. Percentage of each quadrant is shown (24 hours, blue; 48 hours, red). Representative results from three independent experiments are shown here. D, Cell growth in drug-free medium after prior incubation with ATRA, MRT, or ATRA+MRT for 24 and 48 hours (n = 4). Fold change in cell number was calculated by dividing the values at each timepoint with the values at 0d. E, Alteration of CD38 and CD11b expression in HL-60 cells. Surface phenotypes were determined at the indicated timepoints after replating in drug-free medium following incubation with ATRA or ATRA+MRT for 24 hours. Representative results from three independent experiments are shown here. F, Cytoplasmic dsDNA in HL-60 cells 18 hours after combined or single treatment with 1 µmol/L ATRA and 1 µmol/L MRT. Data represent the mean ± SD from three independent experiments. Single data of non-treated cells are shown as a basement level of cytoplasmic dsDNA. G, Cell proliferation of shControl-, shSTING-, and shAIM2-transduced HL-60 cells in drug-free medium after 24 hours pre-exposure to ATRA+MRT (n = 4). Fold change in cell number was calculated by dividing the values at each timepoint with the values at 0d. H, Expression of CD11b on shControl- and shAIM2-transduced HL-60 cells after ATRA+MRT treatment for 24 hours. Representative results and mean ± SD of mean fluorescent intensities (MFI) from three independent experiments are shown here. I, Expression of CD11b on HL-60 cells after ATRA+MRT treatment in the presence of 100 µmol/L Z-WEHD-FMK (BioVision) or DMSO (control) for 24 hours. Representative results and mean ± SD of MFI from three independent experiments are shown here. **, P < 0.01; *, P < 0.05 using two-sided Student t test. d, day.