Liver-specific Phb1 KO mouse (C57BL/6J) was developed by serial breeding of Phb1loxP/loxP and Albumin-Cre+/+(Alb-Cre+/+) mice as shown in Supporting Fig. 1 and described in detail in Supporting Methods. All experiments were reviewed and approved by the Institutional Animal Care and Use Committee
at the University of Southern California. Mice aged between 3 and 46 weeks were used for the experiments. Please see Supporting Methods for details of specimen handling. Isolated hepatocytes were obtained by the Cell Culture Core of the USC Research Center for Liver Diseases as described.14 A normal mouse hepatocyte cell line, AML12, was purchased from American Type Culture Collection (ATCC, Manassas, VA), whereas HepG2 and Huh-7 cells were provided by the Cell Culture Core and cultured in recommended media RAD001 manufacturer in a humidified incubator at 37°C and CO2 at 5%. Cells with passage number <18 were used for the experiments. Primary human hepatocytes were obtained from CellzDirect (Pittsboro, NC). Cells were washed with phosphate-buffered mTOR inhibitor saline three times and protein was extracted for western
blot analysis as described below. The predesigned small interfering RNA (siRNA) targeting mouse Phb1 (sense sequence: AGAGCGAGCGGCAACAUUUtt or AGAAACCAAUUAUCUUUGAtt) and negative control siRNA were purchased from Ambion (Austin, TX). AML12 and Huh-7 cells in six-well plates (0.2 × 106 cells/well); the cells were transfected using RNAiMax (5 μL/well) from Invitrogen (Carlsbad, CA) with PHB1 siRNA (12 nM) or negative control siRNA for 18 hours (AML12) and 48 hours (Huh-7) for mRNA or protein expression or 24 hours (AML12) and 48 hours (Huh-7) for proliferation or apoptosis assays, following the manufacturer’s manual. Phb1 overexpression vector (PHB1-pcDNA3.1) and negative
control empty vector were kindly provided by Dr. Mehta (Illinois Institute of Technology Research Institute, Chicago, IL). Transient transfection was done using 3 μL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and 1.4 μg of target plasmid per well of six-well plates. After 4 hours, the transfection medium was changed to normal medium and cells were cultured for an additional 44 hours for mRNA, protein expression, proliferation, or apoptosis assays. Genomic DNA for genotyping was isolated from hepatocytes and various organs by the method Amino acid of Strauss.15 Total RNA was isolated from livers, AML12, and Huh-7 cells using Trizol reagent (Invitrogen) and then purified by total RNA isolation kit (Bioland Scientific LLC, Cerritos, CA) following the manufacturer’s manuals. Genotyping was determined by polymerase chain reaction (PCR) and is described in detail in Supporting Methods. Northern blot analysis, autoradiography and densitometry were done as described.12 The specific probes for mouse Phb1 exon 2 and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) were designed to correspond to published mRNA sequences from +52 to +154 (Phb1, NM_008831.