Activation of Nrf2 in Atg7-deficient hepatocellular adenoma. (A) Immunoblot analysis. Total, soluble, and insoluble fractions were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Note that the decrease in conversion of LC3-I to LC3-II, which signifies defective autophagosome formation, was confirmed in both the nontumor and tumor regions of the liver of Atg7-deficient mice (Atg7^f/f;Alb-Cre). Data were obtained from three independent experiments. (B) Immunofluorescence analysis. Liver nontumor or tumor sections from 12-mo-old mice of the indicated genotypes were immunostained with anti-p62 and anti-Keap1 antibodies. The right columns show the merged images of Keap1 (green) and p62 (red). Each inset is a magnified image of the boxed region. Bars, 20 µm. (C) qRT-PCR analysis. Total RNAs were prepared from liver or tumor shown in A. Values were normalized to the amount of mRNA in the Atg7^f/f liver of 4-mo-old (left) and 12-mo-old mice (right). Data are means ± SD of three experiments. **, P < 0.01; ***, P < 0.001.

Deletion of p62 suppressed anchorage-independent growth of human HCC cell line. (A) qRT-PCR analyses of p62 and Nqo1 in HCC cell lines. Values were normalized to the amount of mRNA in Hek293 cells. Data shown are representative of three separate experiments. (B) Immunoblot analysis. Total lysates from the indicated cell lines were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Asterisk indicates a nonspecific band. Data were obtained from three independent experiments. (C) Immunoblot analysis of nuclear Nrf2. Nuclear fractions were prepared from the indicated cell lines, subjected to SDS-PAGE, and analyzed by immunoblotting with antibodies against Nrf2 and lamin B (as control). Data were obtained from three independent experiments. (D) qRT-PCR analysis. Total RNAs were prepared from JHH-5 and JHH-5_p62^−/− cells. Values were normalized to the amount of mRNA in JHH-5 cells. (E) Immunoblot analysis of p62. Total lysates from JHH-5 and JHH-5_p62^−/− cells were subjected to SDS-PAGE and analyzed by immunoblotting with p62 and actin antibodies. (F) Double immunofluorescence microscopy. The indicated cell lines were immunostained with anti-p62 and anti-Keap1 antibodies. Bottom row: merged images of p62 (green) and Keap1 (red). Each inset is a magnified image. Bars, 10 µm. (G) qRT-PCR analyses of Nrf2 target genes under three-dimensional (3D) culture conditions. Total RNAs were prepared from JHH5 and JHH-5_p62^−/− cells under 3D culture condition. Values were normalized to the amount of mRNA in the JHH-5. Data are means ± SD of three experiments. ***, P < 0.001. (H) Soft agar colony formation assay. The JHH-5, JHH-5_p62^−/−, and wild-type p62- or p62 mutant (p62 T350A)–introduced JHH5_p62^−/− cells were tested for their ability to grow in soft agar. The transformation was determined according to the protocol supplied by the manufacturer. RFU, relative fluorescent unit. Data are means ± SD of three experiments. *, P < 0.05; ***, P < 0.001.

Loss of Atg7 eventually triggers hepatocellular adenoma. (A) Chronological changes in gross anatomy of livers of Atg7^f/f;Alb-Cre and Atg7^f/f mice. Arrows indicate microtumors. Bars, 1 cm. (B) H&E-stained liver sections of Atg7^f/f and Atg7^f/f;Alb-Cre mice aged from 4 to 12 mo. Dashed line: the tumor region in the liver of a representative 12-mo-old Atg7^f/f;Alb-Cre mouse. The inset shows higher magnification view of the boxed region containing a mitotic cell (arrowhead). C, central vein; P, portal vein. Bars, 100 µm. (C) Liver function tests of Atg7^f/f (blue bars) and Atg7^f/f;Alb-Cre (red bars) mice aged from 4 to 12 mo. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. Data are means ± SD of Atg7^f/f (n = 3) and Atg7^f/f;Alb-Cre (n = 4) at 4 mo of age, of Atg7^f/f (n = 3) and Atg7^f/f;Alb-Cre (n = 3) at 9 mo of age, and of Atg7^f/f (n = 5) and Atg7^f/f;Alb-Cre (n = 5) at 12 mo of age. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Electron microscopy of nontumor hepatocytes (a and b) and tumor cells (c and d) in Atg7^f/f;Alb-Cre mouse liver. The boxed regions in a and c are enlarged and shown as b and d, respectively. The inset in b shows the higher magnification view of a peroxisome (boxed region in b). Note numerous peroxisomes (asterisks) and accumulation of deformed mitochondria (arrowheads) in the peripheral area. Arrows: concentric membranous structures that were consistently present in Atg7-deficient hepatocytes. Nu, nucleus; L, lipid droplet. Bars: (a and c) 5 µm; (b and d) 1 µm. (E) Histochemical detection of COX activity in liver sections of Atg7^f/f and Atg7^f/f;Alb-Cre mice aged from 4 to 12 mo. Dashed line: liver tumor region in a representative 12-mo-old Atg7^f/f;Alb-Cre mouse. Bar, 10 µm. (F) Phospho-histone H2A.X-positive cells in nontumor and tumor regions of 12-mo-old mice of the indicated genotypes. Arrows, cells positive for Phospho-histone H2A.X. Bar, 100 µm.

Activation of Nrf2 in human HCC containing p62- and Keap1-positive aggregates. (A) Immunohistochemical images. Paraffin sections of a liver cancer tissue array were stained with anti-p62 or anti-Keap1 antibody. 6 out of 26 HCC cases containing the typical inclusions are shown. Case codes with pathological grade in parenthesis are indicated at the top. Higher magnification views of the boxed regions are shown in the insets. Bars: (panels) 50 µm; (insets) 10 µm. (B) Double immunofluorescence microscopy. Three cases of HCC containing typical inclusions were immunostained with anti-Keap1 and anti-p62 antibodies. The merged images of Keap1 (green) and p62 (red) are shown on the right. Pathological grades are indicated on the left. Bars, 10 µm. (C) qRT-PCR analyses. Total RNAs were prepared from both nontumor and tumor regions of 15 HCCs. Values were normalized to the amount of mRNA in the nontumor region of B6 where p62- and Keap1-positive aggregates were not recognized. Data are means ± SD of three experiments.