Fig. 1.

HNF4α is heterogeneously expressed in HCC. a Immunofluorescence (IF) reveals P1/P2-HNF4α expression and subcellular localization in mouse and human HCC, hepatoblastoma cancer lines, and in the nontransformed liver cell line, AML12, grown in monolayer conditions. b RT-PCR reveals mRNA abundance of P1/P2-HNF4α in HCC cell lines in vitro, fold change over AML12 Hnf4a mRNA. Compared to ZT0 at the same time, ^*P < 0.01, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, one-way ANOVA test, Dunnett’s multiple comparisons test. (N = 4). c Western blot showing P1/P2-HNF4α abundance in whole cell lysates of cancer cell lines and nontransformed AML12 cells. d Staining of 3D, HCC- and hepatoblastoma-derived spheroids from HepG2 and Hepa-1c1c7 cells with antibodies against P1/P2-HNF4α. e IF staining of spontaneous HCC isolated from jet-lagged WT mice using antibody to P1/P2-HNF4α. f mRNA expression of HNF4a in human HCC tumors and surrounding normal liver tissue (R2: genomics analysis and visualization platform). g IF staining of human normal (“Ctrl”), cirrhotic, hyperplastic, and HCC tumor tissue using antibody against P1/P2-HNF4α (G = grades, advancement increasing with number). h RT-PCR reveals the circadian expression of P1/P2-Hnf4a, Dbp, Ccnd1, Ccnb1 and Myc following the application of scrambled (Sc) or siRNA specific to P1/P2-Hnf4a in AML12 cells (top panel) or in the liver of WT and HNF4α knockout (KO) mice (bottom panel). Compared to controls at the same time, ^*P < 0.03, ^**P < 0.005, ^***P < 0.001, ^****P < 0.0001, two-way ANOVA test, Sidak’s multiple comparisons test. (N = 8–43). Scale bar is 50 µm. (See Supplementary Table 1 for JTK_Cycle Rhythmicity Statistics.) Error bars = SEM