Hepatocyte Culture and Transfection Protocols

Primary hepatocytes were isolated from C57B6/L mice and cultured in DMEM with 10% FBS as previously described [48 (link), 50 (link)–52 (link)]. FLAG-tagged Hes5 [53 ], LIGHT promoter-luciferase constructs [54 (link)], and MYC-tagged SIRT1 [55 (link)] have been described previously. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine LTX (for DNA plasmid) or Lipofectamine RNAiMax (For siRNA) per vendor recommendation. Luciferase activities were assayed 24-48 hours after transfection using a luciferase reporter assay system (Promega) as previously described [56 (link)–58 (link)].