Figure 5.

Involvement of FoxO1 in mediating the effect of PKR on IRS2. Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (control) or siRNA of PKR (A and B) or siRNA of FoxO1 (D) for 24 h, and the transfected cells were cultured in regular media for another 24 h. The forward transfection of empty vector pCMV6-XL5 (pCMV6) or plasmid containing PKR cDNA sequence (pCMV6-hPKR) was performed, and the cells were then treated with OA (2 nM) or its vehicle, ethanol, as a control, for 1 h (C). Reverse transfection of scramble siRNA (negative control, lanes 1 and 2) or siRNA of FoxO1 (siPKR, lanes 3 and 4) was performed, followed by the forward transfection of empty vector pCMV6-XL5 (pCMV, lanes 1 and 3) or the plasmid containing PKR cDNA sequence (hPKR, lanes 2 and 4; E). After treatment, the cells were harvested, and Western blot analysis was performed to detect the total and phosphorylated levels of FoxO1 (A and C), the total levels of IRS1, IRS2, FoxO1 and β-actin (D and E), and the total and phosphorylated levels of PKR (E). PP2A activity assay was performed to detect the phosphatase activity of PP2A (B). The phosphorylations of FoxO1 at Ser256 normalized to the total FoxO1 protein levels (C) and the protein levels of IRS1 and IRS2 normalized to β-actins (E) are expressed as the average of three samples ± SD from three independent experiments. Student's t test was performed, and p values were calculated for analyzing the differences between the indicated samples.