Guidelines for Care and Use of Laboratory Animals were followed during the investigation. All animal procedures were approved by the Ethical Commission of the University of Cagliari and the Italian Ministry of Health. Male Fischer F-344 rats (100-125 g) purchased from Charles River (Milan, Italy) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (γNull) were originally by Jackson Laboratories (Bar Harbor, Maine, USA). Animals were kept on a laboratory diet (Ditta Mucedola, Milan, Italy) and given food and water ad libitum with a 12-hour light/dark daily cycle.
HCC was induced according to the Resistant Hepatocytes (R-H) model [7 (link)]. Rats were injected intraperitoneally with the chemical carcinogen diethylnitrosamine (DENA, Sigma, MO) at a dose of 150 mg/kg body weight. After a 2-week recovery, rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF, Sigma, MO) for 1 week followed by a two-thirds partial hepatectomy (2/3 PH), and an additional week of 2-acetylaminofluorene diet. The animals were then returned to the basal diet and euthanized at 14 months (Supplementary Figure 1 ). Rats exposed to 2-AAF and 2/3 PH, but in the absence of carcinogen, were used as controls.
HCC was induced according to the Resistant Hepatocytes (R-H) model [7 (link)]. Rats were injected intraperitoneally with the chemical carcinogen diethylnitrosamine (DENA, Sigma, MO) at a dose of 150 mg/kg body weight. After a 2-week recovery, rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF, Sigma, MO) for 1 week followed by a two-thirds partial hepatectomy (2/3 PH), and an additional week of 2-acetylaminofluorene diet. The animals were then returned to the basal diet and euthanized at 14 months (