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. 2012 Oct 16;287(50):42053–42063. doi: 10.1074/jbc.M112.415968

FIGURE 5.

FIGURE 5.

Liver-specific deletion of Ei24 causes hepatomegaly and autophagy defects. A, gross anatomical views of representative livers from Ei24flox/+; Alb-Cre and Ei24flox/flox; Alb-Cre mice at 3 months of age. Livers in Ei24flox/flox; Alb-Cre mice are enlarged. B, graphs showing net weight of livers and ratio of liver/body weight in control and mutant mice. Mean ± S.E. of 3 mice is shown. C, H&E-stained liver sections from control and mutant mice show disorganized hepatic lobules and swollen hepatocytes in Ei24flox/flox; Alb-Cre mice. Insets show higher magnification views. Bar, 50 μm. D, gross anatomy of livers from Ei24flox/flox; Alb-Cre mice at 10 months of age demonstrates the development of multiple tumor-like protrusions (arrows). E, H&E staining of liver sections from Ei24flox/flox; Alb-Cre mice at 10 months of age (first panel). The arrow indicates a tumor-like protrusion. Bar, 100 μm. Higher magnification images of livers from controls (second panel) and Ei24-deficient mice (third panel, protrusion region) at 10 months of age. Bar, 20 μm. F, accumulation of LC3-II, p62 and polyubiquitinated proteins in livers from Ei24flox/flox; Alb-Cre mice. Total liver and spleen proteins were extracted, separated by SDS-PAGE, and analyzed by immunoblotting with anti-LC3 and anti-p62 antibodies. Detergent-soluble and detergent-insoluble fractions from liver homogenates were immunoblotted with anti-ubiquitin and anti-p62 antibodies. G, coimmunostaining with anti-p62 (red) and anti-ubiquitin (green) antibodies reveals that p62 and ubiquitin-positive aggregates are absent from control liver sections, but accumulate heavily in liver sections of Ei24flox/flox; Alb-Cre mice. p62 aggregates colocalize with ubiquitin-positive inclusions in mutant liver. Bar, 10 μm. H, immunoblotting with anti-Nqo1 antibody shows that the Nqo1 protein level is dramatically elevated in Ei24flox/flox; Alb-Cre mice. Levels of Nrf2 remain unchanged in control and Ei24-deficient liver. I, Gstm1, Nqo1, and Cyp2a5 mRNA levels are increased in Ei24-deficient mice. The Gstm1, Nqo1, and Cyp2a5 transcription levels were normalized to actin mRNA. Results are representative of at least three experiments.