Fig. 2

Inverse expression of HNF4α and BMAL1 in HCC. a RT-PCR reveals ARNTL (BMAL1) mRNA abundance in HCC and hepatoblastoma lines expressing varying levels of P1/P2-Hnf4a mRNA as well as the nontransformed hepatocyte cell line, AML12. Levels of BMAL1 mRNA expression compared to AML12 cells, ^**P < 0.001, ^****P < 0.0001, one-way ANOVA test, Dunnett’s multiple comparisons test. (N = 4). b Western blot reveals BMAL1, P1/P2-HNF4α, and CCND1 protein levels in HNF4α-positive and HNF4α-negative liver cancer lines as well as in nontransformed AML12 cells. c Staining of 2D HCC cells and AML12 cells with antibodies to BMAL1 and to P1/P2-HNF4α. Overlap with DAPI nuclear stain. d Human HCC microarray datasets reveal inverse gene expression of HNF4a and ARNTL (BMAL1) mRNA in HCC specimens (P < 0.0017) (N = 134). e Staining of 3D spheroids generated from HepG2 and Hepa-1c1c7 cells with antibody to P1/P2-HNF4α and BMAL1. Overlay with DAPI nuclear signal. f Staining of spontaneous mouse HCC from jet-lagged mice using antibodies to BMAL1 and P1/P2-HNF4α. Overlay with DAPI nuclear stain. g Human HCC specimens stained with antibodies for BMAL1 and P1/P2-HNF4α. Overlay with DAPI nuclear stain (G = tumor grade, increasing with number). Scale bar is 50 µm. Error bars = SEM