FIGURE 1.

Transcription factor ZBTB3 is increased in intrahepatic nerve fibers of livers in patients with HBL. (A) Left: Results of RELI algorithm showing that ZBTB3 binds to CEGRs/ALCDs. The location and sequence of the ZBTB3 binding site within the CEGRs/ALCDs are shown. Right: Levels of ZBTB3, SRCAP, ZBTB4, and ZBTB38 mRNAs were examined in a fresh biorepository of HBL samples (values presented as means±SEM). (B) ZBTB3 and SRCAP protein levels were determined by western blotting. Background (Back) is a section of the normal liver adjacent to the tumor. Hepatoblastoma is a tumor section of the liver. The western blot was performed with backgrounds of 6 patients and with tumor sections of 9 patients with HBL patients. Student t tests were utilized to compare ratios of signals of ZBTB3 (p=0.0009) and SRCAP (p=0.0006) proteins to signals of β-actin (right images). (C) Co-IP studies of ZBTB3 from the background of 3 patients and tumor sections of 4 patients with HBL, as well as from HepG2 cells (labeled G2 on image). Top: Western blot shows the input of the proteins. Ratios of signals of ZBTB3 and SRCAP proteins to signals of β-actin are shown below each western blot image. (D) Left: Pull-down assay with 18BP oligomer from CEGRs/ALCDs containing a binding site for ZBTB3. (B: Background, T: Tumor). Right: ChIP assay with CEGRs/ALCDs of CTNNB1, NFE2L2, and PGAP1 genes using chromatin solutions from HepG2 cells and from patients with HBL. (E) β-III-tubulin staining in background and tumor sections of HBL. (F) Costaining of a large region of HBL with DAPI, β-III tubulin, and ZBTB3. Scale bar=50 μM. Arrows show colocalizations of signals around the bile duct. Circles show colocalizations far away from the bile duct. Abbreviations: CEGRs/ALCDs, cancer-enhancing genome regions/aggressive liver cancer domains; ChIP, chromatin immunoprecipitation; Co-IP, coimmunoprecipitation; HBL, hepatoblastoma; PV-IHBD, portal vein-intrahepatic bile duct area.