FIG 7.

Sirt2-mediated upregulation of HBV replication is independent of HBx. (A) Overexpression of Sirt2 increases replication of a HBx-deficient mutant via AKT/GSK-3β/β-catenin signaling. HepG2 cells were mock transfected (lane 1) or transfected with 4 μg of 1.3-mer HBV WT (ayw) (lanes 2 and 3) or with 4 μg of the 1.3-mer HBx-deficient (ayw) mutant (lanes 4 and 5) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of the 3×FLAG-Sirt2.1 (4 μg) construct. Lysates were prepared at 72 h posttransfection. The amount of transfected DNA was adjusted using pcDNA3 (lanes 2 and 4). SDS-PAGE and Western blotting of proteins (1st to 11th panels), native agarose gel electrophoresis and Western blotting for core particles (12th panel), and Southern blotting of HBV DNA (bottom panel) were performed as described in the legend of Fig. 1. (B) Activation of the AKT/GSK-3β/β-catenin signaling pathway by HBx and Sirt2 is not synergistic. HepG2 cells were transfected with 4 μg of HA-HBx (lane 2), 4 μg of the 3×FLAG-Sirt2.1 construct (lane 3), or 4 μg of HA-HBx plus 4 μg of the 3×FLAG-Sirt2.1 construct (lane 4). Lysates were prepared at 72 h posttransfection. The amount of transfected DNA was adjusted using pcDNA3 (lanes 2 and 3). SDS-PAGE and Western blotting of protein (1st to 11th panels) were performed as described in the legend of Fig. 1. Relative levels of total and active AKT (pT308 and pS473), total/phosphorylated (S9) GSK-3β, and β-catenin were measured using ImageJ 1.46r. Data are presented as mean values from three independent experiments. Statistical significance was evaluated using Student's t test. P values relative to the respective controls are shown.