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. 2009 Dec 14;285(8):5594–5605. doi: 10.1074/jbc.M109.068346

FIGURE 9.

FIGURE 9.

GILZ inhibition of FOXO3 activity requires a FOXO3 functional NES sequence. A, HL-60 cells were transiently transfected with pEGFP-FOXO3-TM plasmid (10 μg) and pDsRed-GILZ (10 μg). After overnight expression, cells were treated with or without 5 ng/ml of leptomycin B, maintained at 37 °C, and analyzed by confocal microscopy for 30 min. The images presented were acquired at 30 min after leptomycin B addition. B, schematic representation of FOXO3-TM-NESm. C, HL-60 cells were transiently transfected with pEGFP-FOXO3-TM-NESm plasmid (10 μg) and pDsRed-GILZ (10 μg) or pcDNA3-Myc. After overnight expression of exogenous proteins, cells were fixed in paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were analyzed by fluorescence microscopy. D, HL-60 cells were transiently transfected with the reporter plasmid p3xIRS-MLP-Luc, with either 0.5 μg of pcDNA3-FOXO3-TM or pcDNA3-FOXO3-TM-NESm, 5 μg of pcDNA3-Myc-GILZ or pcDNA3-Myc, and the transcriptional activity was analyzed after 24 h of culture. Results are expressed as the percentage of p3xIRS-MLP-Luc activity, with 100% representing the activity of the construct in the presence of FOXO3-TM. Data represent the mean ± S.E. of three independent experiments performed in duplicate. *, p < 0.05. Error bars, S.E.