FIGURE 4.

Effects of activation or inhibition of PI 3-Kinase on FoxO1-EGFP localization. A, MIN6 cells, after 24 h of transient transfection with pFoxO1-EGFP (1.0 μg), and either empty vector (pcDNA3; Con) or plasmids encoding p100.CAAX or Δp85 (1.0 μg) were incubated with further additions (as shown, LY294002, 50 μm) for 1 h in KRB medium. B, quantitation of the ratio (mean ± S.E., n = 20–30 cells from ≥ 3 separate experiments) as shown in A. C, effects of glucose and insulin on FoxO1-EGFP localization are mimicked by an active form of protein kinase B but do not require p70 S6 kinase or glycogen synthase kinase-3 (GSK3). Cells were transfected with pFoxO1-EGFP plus either empty vector (pcDNA3; Con), PKB-DD or PKB-myr. After 16 h of incubation in DMEM containing 3 mm glucose, the medium was replaced with medium containing the specified concentrations of glucose, insulin, and GSK3 inhibitors, SB216763 and SB415286 (SB21, SB41, 3 μm and 30 μm, respectively), rapamycin, 30 nm. D, quantification of ratio (mean ± S.E., n = 20–40 cells, obtained from ≥3 independent experiments for each condition). Scale bar, 5 μm.