Figure 6.

Foxo3a luciferase reporter assay. The modulation of the Akt/PI3K/Foxo3a pathway by the cyclotide [T20K]kB1 binding to 14-3-3 proteins was investigated. HEK293 cells were transiently transfected with Foxo3a-Luc plasmid encoding luciferase under a promotor with Foxo response elements, Foxo3a and 14-3-3β. For control the reporter plasmid (basal value) was also co-transfected with the single functional effector proteins 14-3-3β or Foxo3a. Cells were treated under starving conditions with peptide or fusicoccin-A (FC-A) for 4 h or 24 h, respectively. The 14-3-3 stabilizer compound fusicoccin-A is reported to impact the cellular location of 14-3-3 complexed transcription factors (shown by an independent experiment). Wortmannin (Wort.) and LY294002 (LY) served as positive control compounds inhibiting the phosphoinositol 3-kinase and thus interfering with the nuclear export of Foxo3a. IC₅₀-values were calculated of two independent experiments using nonlinear regression with curve fitting and constrains to the top and bottom values. Statistics show one-way ANOVA analysis with Bonferroni Post-Hoc test; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ^****P < 0.0001, n.s., not significant.