Knockdown of FOXO3A suppresses ATRA-induced TRAIL expression, granulocytic differentiation, and cell death of NB4 cells. (A) SCR or FOXO3A shRNA clones were incubated with 1.0μM ATRA (closed) or 0.1% ethanol (open) for the indicated time periods. Cell growth was assessed by the CellTiter-Glo Luminescent Cell Viability Assay Kit. Results are presented as the mean ± SE of 3 independent experiments relative to the results obtained at day 0. SCR (circles), A2 (squares), B4 (triangles), D6 (diamonds). *P < .05: A2 (days 2, 4, and 5), B4 (day 6), D6 (days 4 and 5); **P < .01: A2 (day 6), B4 (day 5), D6 (day 6), compared with SCR with ATRA. Granulocytic differentiation was determined by an NBT reduction assay (B) and CD11b expression (C) by FACS analysis. (D) Morphologic changes were examined by Wright-Giemsa staining and use of an Olympus BX51. Scale bar represents 10 μm. (E) Annexin V–positive cells were detected by FACS analysis. Results of panels B, C, and E are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with SCR; SCR (), A2 (░), B4 (), D6 ().