The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity
J. Clin. Invest. Jun Nakae, et al. 116:2473 doi:10.1172/JCI25518 [Go to this article.]

Figure 3
Effects of 3A FoxO1 and 3A/LXXAA FoxO1 mutants on endogenous Igfbp-1 or G6Pase gene expression in SV40-transformed hepatocytes. (A) Expression of HA-WT, Flag 3A FoxO1, or 3A/LXXAA FoxO1 in SV40-transformed hepatocytes was detected. Lane 1, hepatocytes transduced with adenovirus encoding LacZ; lane 2, with WT; lane 3, with 3A mutant; lane 4, with 3A/LXXAA mutant at 10 MOI. Endogenous Igfbp-1 (B) or G6Pase (C) gene expression in SV40-transformed hepatocytes transduced with adenovirus encoding LacZ, WT FoxO1, 3A FoxO1, or 3A/LXXAA FoxO1. Hepatocytes transduced with each adenovirus were incubated in serum-free medium overnight before addition of dexamethasone/8-Br-cAMP/IBMX and incubation for 8 hours. Isolation of total RNA and real-time PCR were performed as described in Methods. Data (mean ± SEM) are from 3 independent experiments and are normalized by the amount of β-actin mRNA and expressed relative to the corresponding LacZ value. *Statistically significant difference between 3A- and WT- or 3A/LXXAA-transduced cells at each MOI (P < 0.001 by 1-way ANOVA). (D) Effects of 3A FoxO1, 3A/LXXAA FoxO1, and Δ256 FoxO1 mutants on endogenous Igfbp-1 gene expression in SV40-transformed hepatocytes. Hepatocytes transduced with each adenovirus at 10 MOI were incubated in serum-free medium overnight before addition of dexamethasone/8-Br-cAMP/IBMX for 8 hours. Isolation of total RNA and real-time PCR were performed as described in Methods. Data are from 3 independent experiments and are expressed as mean ± SEM of fold of relative gene expression for β-actin in LacZ-transduced hepatocytes in the absence of dexamethasone/8-Br-cAMP/IBMX.