Research Paper Volume 4, Issue 7 pp 462—479

Figure 7. Methylation of FoxO3 at lysine 271 promotes FoxO3 degradation, while increasing FoxO3 transcriptional activity. (A) Methylated FoxO3 is less stable than the unmethylated form. 293T cells were co-transfected with Flag-FoxO3 and Flag-Set9 (WT or H297A). The cells were then treated with MG132 (20 μM), ALLN (40 μM) or PSI1 (40μM) for 16 hours prior to lysis. Extracts were analyzed by western-blot with antibodies to K271me1, Flag, c-Jun, and b-actin. (B) Western-blots from (A) were analyzed by densitometry and the ratio of intensity in the presence or absence of proteasome inhibitor was compared after normalization to b-actin. * p< 0.05, **p<0.01, One way ANOVA, Bonferroni post test. (C) K271R is more stable than wild type FoxO3. 293T cells were transfected with Flag-FoxO3 (WT or K271R) together with Flag-Set9 (WT or H297A). Cells were treated for the indicated length of time with 20 μM cycloheximide (CHX) and FoxO3 expression was tested by western-blot with antibodies to Flag. (D) Western-blots from (C) were analyzed by densitometry at each time point of cycloheximide treatment. Results were normalized to the untreated well for each condition. (E) Set9 increases the transcriptional activity of the constitutively nuclear form of FoxO3. Flag-Set9 (WT or H297A) was co-expressed with Flag-FoxO3 (WT, K271R, TM [T32A/S253A/S315A], or TM K271R [T32A/S253A/K271R/S315A]) in 293T cells together with a 6xDBE-luciferase reporter construct, and luciferase activity was measured. The data represents the mean and SEM from 5 independent experiments, conducted in triplicate. All samples were normalized to the Flag-FoxO3 WT + Flag-Set9 WT condition. * p< 0.05, ** p< 0.01, *** p< 0.001, ns: p> 0.05, One way ANOVA, Bonferroni post test.