Figure 1
Figure 1. ATRA induces dephosphorylation of FOXO3A and translocation of FOXO3A into the nucleus. (A) NB4 cells were incubated with 1.0μM ATRA or a final volume of 0.1% ethanol for the indicated time periods. Total protein was extracted and analyzed by Western blotting using antibodies for the indicated antigens. β-actin was used as an internal control. (B) NB4 cells were transfected with empty vector (pcDNA3) or dominant negative form of AKT (DN-AKT) by electroporation. After incubation for 48 hours, the cells were harvested. Total protein was extracted and submitted to Western blotting with the indicated antibodies. (C) NB4 cells or APL blast cells from patients were incubated with 1.0μM ATRA or a final volume of 0.1% ethanol for 48 hours. After fixation and permeabilization, cells were incubated with anti-FOXO3A antibody and Alexa488-conjugated goat anti–rabbit IgG (green). The 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. (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. Results are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with the ethanol control. (E) After cross-link reversal, the recovered DNA was subjected to ChIP analysis with FOXO3A antibody or normal serum IgG (as a control) using conventional PCR. The presence of promoter DNA before immunoprecipitation was confirmed by PCR (input). NTC refers to the no-template control, which lacked input DNA. (F) TRAIL promoter activity, as determined by measurement of luciferase activity, was assayed in NB4 cells transfected with the indicated constructs after a 48-hour incubation with 1.0μM ATRA (■) or a final volume of 0.1% ethanol (□). mt1, mt2, and mt3 represent the human TRAIL promoter gene that had a mutated proximal FOXO3A binding site, a mutated distal FOXO3A binding site, or both mutated sites. Results are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with empty pGL2m vector-transfected control (Vec).

ATRA induces dephosphorylation of FOXO3A and translocation of FOXO3A into the nucleus. (A) NB4 cells were incubated with 1.0μM ATRA or a final volume of 0.1% ethanol for the indicated time periods. Total protein was extracted and analyzed by Western blotting using antibodies for the indicated antigens. β-actin was used as an internal control. (B) NB4 cells were transfected with empty vector (pcDNA3) or dominant negative form of AKT (DN-AKT) by electroporation. After incubation for 48 hours, the cells were harvested. Total protein was extracted and submitted to Western blotting with the indicated antibodies. (C) NB4 cells or APL blast cells from patients were incubated with 1.0μM ATRA or a final volume of 0.1% ethanol for 48 hours. After fixation and permeabilization, cells were incubated with anti-FOXO3A antibody and Alexa488-conjugated goat anti–rabbit IgG (green). The 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. (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. Results are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with the ethanol control. (E) After cross-link reversal, the recovered DNA was subjected to ChIP analysis with FOXO3A antibody or normal serum IgG (as a control) using conventional PCR. The presence of promoter DNA before immunoprecipitation was confirmed by PCR (input). NTC refers to the no-template control, which lacked input DNA. (F) TRAIL promoter activity, as determined by measurement of luciferase activity, was assayed in NB4 cells transfected with the indicated constructs after a 48-hour incubation with 1.0μM ATRA (■) or a final volume of 0.1% ethanol (□). mt1, mt2, and mt3 represent the human TRAIL promoter gene that had a mutated proximal FOXO3A binding site, a mutated distal FOXO3A binding site, or both mutated sites. Results are presented as the mean ± SE of 3 independent experiments. **P < .01 compared with empty pGL2m vector-transfected control (Vec).

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