Caspase-6– and -8–mediated myeloid differentiation is abrogated by AML-associated cytoplasmic NPM mutant. OCI/AML-3 cells were transfected with either specific (siNPM) or control (siNEG) siRNA with or without exposure to 10μM caspase-6 or caspase-8 inhibitors. (A) Forty-eight hours after transfection, the cells were subjected to May-Grünwald-Giemsa stain and observed under light microscope (original magnification ×100). Black arrowheads indicate cells differentiated past the promyelocytic stage. Scale bar represents 20 μm. (B) Top: Cells from panel A were examined, and the number of myeloid blasts, granulocytes, and monocytes/macrophages were scored. At least 200 cells were scored for each set of experiments. Bottom: Representative micrographs of a myeloid blast, mature granulocyte, monocyte, and macrophage. (C) Cells from panel A were also analyzed for expression of cell surface markers for myeloid differentiation (CD11b, CD14, and CD33). (D) Transformation-like phenotype of cells treated as in panel A. Cells 3 days after siRNA transfection were plated in methylcellulose medium and incubated at 37°C for 21 days. Top: Quantification of the colonies for each experimental condition. Bottom: Growth of the transfected cells in methylcellulose. Scale bar represents 2.0 mm. (E) HL-60 cells were transfected with caspase-6, -8, -3, or p53, and GFP. In the case of caspase-6 or -8 transfections, a portion of cells were cotransfected with GFP-tagged NPMc instead of GFP. Forty-eight hours later, cells were subjected to May-Grünwald-Giemsa stain and observed. Black arrowheads indicate cells differentiated beyond the promyelocytic stage. Scale bar represents 10 μm. (F) Caspase activities were measured in caspase-6– or -8–transfected cells from panel E. **P < .01. Data were obtained from 3 sets of experiments and presented as mean ± SEM.