Tandem affinity purification of TRIM24 containing complexes. (A) Anti-FLAG immunoblot to detect expression of E-TRIM24 in nuclear extracts from retroviral transduced or control HeLa cells. (B) Detection of E-TRIM24 expression and localization by immunofluorescence with anti-FLAG antibody. (C) SDS-PAGE and silver nitrate staining of tandem affinity purified TRIM24 and its partner proteins. The identity of proteins identified by MS is indicated. (D) Western blot analysis performed on the tandem immunopurified complex and the flowthrough (FT) and bound (B) fractions of the subsequent anti-TRIM33 or TRIM28 immunoprecipitations. (E) Schematic representation of the major TRIM24/TRIM33 and minor TRIM24/TRIM33/TRIM28 containing complexes. (F) Western blot analysis of immunoprecipitates of adult liver extracts. Immunoprecipitations were performed with antibodies against the endogenous TRIM proteins or only with protein G-sepharose beads, as indicated above each lane and the presence of the TRIM24 and TRIM33 proteins in the precipitated fractions visualized by immunoblot.

Characterization of hepatocyte-specific TRIM24 knockout mice. (A) and (B) Western blot analysis with anti-TRIM24 and β-tubulin antibodies performed on liver and testis extracts from Trim24^L2/L2 mice whose genotypes with respect to the Alb-Cre and Alb-CreER^T2 transgenes are shown above each lane. In B all mice were injected with Tam. (C) and (D) Ki67 staining performed on liver sections from 3-wk-old Trim24^L2/L2 mice (C) or 5-mo-old Tam injected Trim24^L2/L2 animals (D) with the indicated transgenes. The inserts in the upper DAPI-stained panels show higher magnification images of enlarged polyploid nuclei. (E) and (F) Quantification of Ki67-positive hepatocytes in the indicated strains. Means (N = 4) ± SD *P < 0.05, **P < 0.005.

Comparison of gene expression in TRIM24 germ line and hepatocyte-specific knockout mice. The expression of six representative genes was determined by RT-qPCR on whole total liver RNA from WT, Trim24^-/- or Trim24^L2/L2 littermates with the indicated transgenes. Expression in the control mice was arbitrarily considered as = 1. Means (N = 4) ± SD *P < 0.05, **P < 0.005.

Spontaneous liver tumor formation in hepatocyte-specific TRIM24 knockout mice. (A–C) Representative macroscopic views of 14-mo-old Trim24^-/- (A), Trim24^L2/L2/Alb-Cre⁺ (B), and Trim24^L2/L2/Alb-CreER^T2+ (C) livers showing multiple tumor nodules, which were histologically classified as HCA or HCC. (D–F) Hematoxilin/Eosin staining of representative histology sections of HCA, the tumor is well circumscribed and causes compression of the surrounding nontumoral parenchyma. (G–I) Histology sections of solid HCC composed of hepatocytes with occasional eosinophilic cytoplasmic inclusions (arrows). (J) Statistics of neoplasias observed in germinal and hepatocyte-specific constitutive and Tam-inducible TRIM24 mutants. Neoplasias are classified as FCA, HCA, or HCC.

Spontaneous HCC tumor formation in hepatocyte-specific TRIM28 and TRIM33 knockout mice. (A) and (B) Western blot analysis of TRIM28 and TRIM33 expression following hepatocyte-specific inactivation. (C) Statistics of macroscopic liver neoplasias in mice with the indicated genotypes. Livers were classified in two groups: Livers containing one to three tumors with a size > 1 mm are indicated by the open bars and livers containing numerous tumors (> 3) are indicated by the filled bars. (D) Macroscopic views of livers from mice with the indicated genotypes. Lesions are indicated by arrows. (E–G) Expression of TRIM24 target genes was quantified by RT-qPCR in all single and double-mutant mice. Expression in the control mice was arbitrarily considered as = 1. Means (N = 4) ± SD *P < 0.05.