Hepg2

MK1^± mice were bred to generate mouse embryonic fibroblasts (MEFs) with or without MKRN1 expression. To obtain MEFs on day 13.5 from the embryos, embryos were minced after being rinsed with phosphate-buffered saline (PBS; Welgene) and incubated with 3 ml trypsin/EDTA (Gibco) at 37 °C for 15 min. MEFs were grown in 20 ml Dulbecco's modified Eagle medium (DMEM; GIBCO) containing 10% fetal bovine serum (FBS; GIBCO) for 4–8 h before the medium was replaced with DMEM containing 10% FBS. The confluent MEFs were subsequently subcultured at a ratio of 1:3. 293T (a cell line derived from human embryonic kidneys) and HepG2 (a cell line derived from human hepatocellular carcinoma) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The phenotypes of these cell lines were authenticated by ATCC on a regular basis. All cell lines used in this study were negative for mycoplasma when detected using an e-Myco plus Mycoplasma PCR Detection Kit (Intron, Gyeonggi-do, Republic of Korea) and were protected from mycoplasma infection by treatment with PlasmocinTM (InvivoGen, CA, USA). To transfect plasmid DNA or short interfering (si)RNA, either PEI (Sigma-Aldrich, St. Louis, MO, USA) or Lipofectamine RNAiMAX (Invitrogen) was utilized.