Knockdown of FOXO3A does not affect ATRA-induced PML-RARα degradation or subsequent formation of NBs in NB4 cells. (A) Total protein was extracted from SCR or FOXO3A shRNA clones cells and submitted to Western blotting with antibodies against the indicated antigens. β-Actin was used as an internal control. (B) SCR or FOXO3A shRNA clones cells were incubated with 1.0μM ATRA or a final volume of 0.1% ethanol for 48 hours. Total protein was extracted and submitted to Western blotting with antibodies against indicated the antigens. β-Actin was used as the internal control. (C) NB4, SCR, or FOXO3A shRNA clones were incubated with or without 1.0μM ATRA for 48 hours and fixed on poly-l-lysine–treated glass slides. After permeabilization, cells were incubated with anti-PML antibody and Alexa488-conjugated goat anti–mouse IgG. Imaging analysis was performed with an Olympus IX70 microscope. Scale bar represents 10 μm. (D) TRAIL mRNA was detected by quantitative RT-PCR using TaqMan probes for TRAIL and GAPDH. Quantification was performed using the relative standard curve method. ■ and □ represent 1.0μM ATRA and 0.1% ethanol, respectively. Results are the mean ± SE of 3 independent experiments. *P < .05 and **P < .01 compared with the SCR control.