Figure 2.

USP7 affects FoxO1 transcriptional activity. A, USP7 suppresses transcriptional activation of FoxO1 on the 3X IRS FoxO1 response element-luciferase reporter construct in HEK293A cells. Data presented as relative activity and shown as ± SEM; n = 3. ***, P < .001 by one-way ANOVA with Newman-Keuls Multiple Comparison test. Effect is noted in absence of changes on FoxO1 protein levels, as indicated by included Western blot analysis of whole-cell extracts. B, Constitutively active Akt (Myr Akt) suppresses FoxO1 transcriptional activity to a further degree than USP7 alone. Data presented as in (A). C, USP7 suppresses transcriptional activation of a noninsulin-sensitive FoxO1 mutant (FoxO1 mut.). Cells were deprived of serum for 6 h prior to harvest. Data presented as relative activity and shown as ± SEM; n = 3. *, P < .05; **, P < .01; and ***, P < .001 by one-way ANOVA with Newman-Keuls Multiple Comparison test. D, USP7 fails to affect FoxO1 nuclear/cytoplasmic localization. Fluorescence microscopy images showing typical nuclear and cytoplasmic localization of FoxO1-GFP. Cells were treated as in (C).