Fig. 3: Preneoplastic cells are derived from hyperpolyploidy-enriched CL region.

a Nucleus and cell size within preneoplastic foci and non-preneoplastic region. n > 400 hepatocytes/group, N = 6 mice/group. b Schema for isolation of PL- and CL-enriched hepatocytes by digitonin-collagenase perfusion system. c Representative images display discoloration pattern on liver surface after digitonin infusion from inferior vena cava or portal vein, respectively. d Representative images show the H&E staining of indicated liver histologically. Digitonin infusion caused discoloration of eosin signal. N = 4 mice/group. e qPCR analysis of mRNA levels of CL- and PL-specific markers. N = 3 mice/group. f The nucleus morphology of hepatocytes isolated by digitonin-collagenase infusion. The DNA contents of CL- and PL-enriched hepatocytes were analyzed by flow cytometry. N = 4 mice/group. g The colony numbers at 21th day in vitro (DIV) in indicated cultured hepatocytes. n = 4 independent experiments. h Representative images of colony and non-colony hepatocytes morphology isolated from CL region at 21th DIV. Low and high magnification show the morphology of colonies and nuclei, respectively. n = 4 independent experiments. i Nucleus size in colony and non-colony hepatocytes at 21th DIV. n = 500 and 257 cells from four independent experiments, respectively. j Visible differences in tumor size under the skin of the dorsal body surface in NSG^TM mice after 10 weeks of subcutaneous transplantation. Arrow and arrow head indicate subcutaneous implantation of hepatic spheres derived from CL- and PL-enriched hepatocytes, respectively. Enlarged tumors were observed in CL-enriched hepatocytes transplanted region in j-2 and -3 mice. No tumors were detected in j-1 mouse. Line graphs show the tumor growth cure of subcutaneous transplants. N = 6 mice. k Example of images show H&E staining of tumor derived from CL-enriched hepatocytes under low and high magnification. Arrows (cyan) indicates the proliferating tumor cells at metaphase. The highest images display bipolar and multipolar dividing cells in tumor nodes. Dashed lines delineate cell borders. N = 6 mice for each implantation. l Line and dot graph show the quantitative data of nucleus size within preneoplastic foci, non-preneoplastic region and tumor derived from CL-enriched hepatocytes. n = 420, 408, and 420 cells, respectively. Statistic: Two-tailed Student’s unpaired t-test (a, i); two-tailed Mann–Whitney nonparametric test (e); one-way ANOVA (g) and (l); two-way ANOVA (j). Values represent the mean ± SEM (also see Supplementary Fig. 3).