Rat liver specimens were obtained from previous experiments. Healthy livers, pools of liver preneoplastic lesions isolated with

the use of a stereomicroscope, and HCC were used. The transplantation procedure was performed as previously described.19, 20 Briefly, diabetes was induced in adult inbred male Lewis rats (250-300 g) by treatment with a single subcutaneous dose of streptozotocin (80 mg/kg body weight [BW]) and was defined by a nonfasting Deforolimus nmr blood-glucose level higher than 400 mg/dL, manifesting between 1 and 3 days after the administration of streptozotocin. Islets of Langerhans were isolated from nondiabetic littermates and transplanted into the liver of recipient rats through the portal vein. A low number of islets (250-450 islet grafts per animal) was transplanted so that mild hyperglycemia (250-300 mg/dL) persisted for at least 10 months after transplantation. During infusion, the branch supplying the left part of the liver was clamped, thus ensuring that the transplants were embolized only into the right part of

the liver and the left part served as an intraindividual control. As an additional control, the livers of nondiabetic rats not undergoing transplantation were used. However, because there were no significant differences see more in the parameters examined between the intraindividual controls and the nondiabetic rats not undergoing transplantation, the data of these two groups were combined and referred to as the control liver. Animals were sacrificed under anesthesia between 2 days and 24 months after transplantation. Groups of rats were subjected to daily administration

of the phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, NVP-BEZ235 (kindly provided by Novartis, Basel, Switzerland), dissolved in 1% methylcellulose at a concentration of 10 mg/kg BW for 4 weeks. Rats were housed, fed, and treated according to the German Animal Protection Law and approved by the Local Government of Mecklenburg-Vorpommern. Transfection of Hep3B and HLE cell lines with short interfering 上海皓元 RNAs (siRNAs) and treatment with specific inhibitors were performed as described in the Supporting Materials and Methods. Hepatic tissue samples were homogenized and processed as previously reported.26 Nitrocellulose membranes were probed with specific primary antibodies (Supporting Table 1). Tukey-Kramer’s test was used to evaluate statistical significance. P < 0.05 was considered significant. Data are expressed as means ± standard deviation (SD). See the Supporting Materials and Methods for detailed descriptions of materials and methods.