Forced expression of active FOXO3A induces apoptosis of NB4/RA cells. (A) Total protein was harvested from vector control (VCont) and FOXO3A-TM-ER clone cells and submitted to Western blotting with antibodies for FOXO3A and β-actin. (B) Cells were incubated with 0.7μM 4-OHT or final volume of 0.07% ethanol for 48 hours and fixed on poly-l-lysine–treated glass slides. After permeabilization, cells were incubated with anti-FOXO3A antibody and Alexa488-conjugated goat anti–rabbit IgG (green). Nuclei were stained with TO-PRO-3 (red). Confocal imaging analysis was performed using a Leica TCS-NT laser confocal microscope. Scale bar represents 10 μm. (C) Cells were incubated with 0.7μM 4-OHT (closed) or a final concentration of 0.07% ethanol (open) at the indicated times. Cell growth was assessed using 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; VCont (circles), no. 13 (squares), no. 16 (triangles), and no. 18 (diamonds). *P < .05: no. 13 (day 1); **P < .01: nos. 13, 16, and 18 at day 2 compared with VCont treated with 4-OHT. (D) Annexin V–positive cells were detected by FACS analysis. (E) Nuclei were stained using Hoechst 33342. Imaging analysis was performed with an Olympus IX70 microscope. Scale bar represents 10 μm. (F) TRAIL mRNA was detected by quantitative RT-PCR using predesigned TaqMan probes for TRAIL and GAPDH. Quantification was performed using the relative standard curve method. (G) Total protein was extracted at 48 hours and blotted. The blots were probed with antibody for TRAIL and were reprobed with anti–β-actin antibody to confirm equal protein loading. Results of panels D and F are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with the VCont treated with 4-OHT.