18 These studies were approved by the Ethical Committee of the Second Military Medical University. The rat pluripotent LPC-like cell line WB-F34419, 20 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, PF-2341066 CA) supplemented with 0.5% fetal bovine serum (FBS; Gibco, Invitrogen). WB-F344 cells treated with TGF-β (Miltenyi Biotec, Auburn, CA) at 0.25 ng/mL or saline for 18 weeks were termed WB-TβLT or WB-CON

cells, respectively. The mouse liver progenitor cell line LEPC was cultured in DMEM supplemented with 10% FBS.21 Adenovirus encoding dominant-negative mutant of Akt (AdDN-Akt) and green fluorescent

protein (AdGFP) were generated using AdMax Adenovirus Vector (Microbix, Ontario, Canada). All human liver tissues were obtained from surgical resections of patients without preoperative treatment at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). (See Supporting Table 1 for detailed clinicopathologic information.) The procedure for human sample collection was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital. Formaldehyde-fixed, paraffin-embedded sections of liver tissue were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry following routine protocols medchemexpress as described.22 PXD101 The antibody information is provided in Supporting Table 2. Frozen sections of fresh human or rat liver

tissue were incubated with rabbit anti-CD133 (Abcam, Cambridge, MA) and mouse anti-OV-6 (R&D Systems, Minneapolis, MN), followed by fluorescent staining with Alexa Fluor 488-conjugated antimouse IgG and Alexa Fluor 555-conjugated antirabbit IgG (Invitrogen). Mice samples were stained by rabbit anti-A6 and FITC-conjugated antirabbit IgG (Invitrogen). Nuclear staining was performed by Hoechst 33342 in tissue samples. Rabbit anti-forkhead family of transcriptional regulators subfamily O, 3a (FOXO3a; Epitomics, Burlingame, CA) and Alexa Fluor 555-conjugated antirabbit IgG were used to detect the cellular localization of FOXO3a in WB-CON and WB-TβLT cells, and 4′,6-diamidino-2-phenylindole (DAPI) was applied to show the nucleus. Representative images were captured with an Olympus IX70. Quantitative PCR was performed using SYBR Green PCR Kit (Applied Biosystems, Foster City, CA) and ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). The messenger RNA (mRNA) level of specific genes was normalized against β-actin. Primers used are listed in Supporting Table 3. Total RNAs were isolated using TriZol (Invitrogen). The level of microRNA (miRNA) was determined using specific primers (RiboBio Biotechnology, Guangzhou, China) and normalized against U6.