Figure 6.

p62 accumulates in non-mutated HCCs. (A) Keap1 mRNA expression levels in surrounding tissue and microdissected HCCs of rats sacrificed 13 months after DENA administration. Relative mRNA expression was calculated by using the 2^-∆∆CT method and Gapdh as endogenous control. Gene expression is reported as fold-change relative to surrounding tissue. Each bar represents mean ± standard error (SEM) of 6 to 9 samples per group. ns; not significant; (B) Microphotograph showing IHC of serial sections displaying no evidence of p62 or KEAP1 positivity in GSTP+ preneoplastic nodules at 4 months after DENA administration (5×); (C) Representative images of IHC of HCCs showing positivity for GSTP, NQO1, KEAP1 and p62 at 10 (top) and 13 (bottom) months after DENA injection (5×); (D) Western blot analysis showing p62, Nrf2, phospho-p62, myc and phospho-S6 levels in HCCs and in surrounding and normal liver. Numbers indicate the band intensity ratio between the protein of interest and the respective housekeeper protein (actin); (E) Co-immunoprecipitation assay. Total lysates from normal livers, surrounding tissues and HCCs were subjected to immunoprecipitation with anti-p62 antibody. The immunocomplexes were examined by immunoblotting with the indicated antibody; (F) Western blot analysis showing p62 levels and LC3 levels in WT and mutated HCCs developed 13 months after DENA treatment and in the surrounding liver tissue; accumulation of p62 is observed in non-mutated (WT) HCCs. Upper numbers indicate the band intensity ratio between the protein of interest and the respective housekeeper protein (actin); lower numbers indicate the band intensity ratio between LC3II and LC3I. See also Figures S7–S9 for detailed information about Figure 6D–F.