Mir 451a mimic

Western blotting was performed in accordance with a previous protocol [18] . Briefly, proteins were loaded on 5-12% tris-acrylamide gels and membranes were blotted with specific antibodies. The LPIN1 antibody (Cat. 14906, Cell Signaling Technology, USA) was diluted to 1:1000. Bands were detected using a Gel imaging system (SYNGENE, USA).
Plasmid construction and transfection SMMC-7721, Hep3B, and HUVECs (3 × 10 5 cells/well) were seeded in 6-well plates, incubated overnight, and then transfected with miR-451a mimics (Ribobio Co., China) using Lipofectamine2000 (Invitrogen) and Opti-MEM (Corning) according to the manufacturer's instruction. Negative control (NC) is also purchased from Ribobio Co.
To construct the human LPIN1 overexpression vector, human LPIN1 cDNA was cloned from SMMC-7721 cells and ligated into pLV-EF1α-MCS-IRES-Bsd (Biosettia Inc., USA). For gene silencing, shLPIN1 DNA oligomers were annealed and cloned into pLV-H1-EF1α-puro vector (Biosettia Inc., USA). These sequences are listed in the supplementary materials section. Scrambled control (SC) sequences were used as in a previous report [16] .

The human HCC cell lines Hep3B and HepG2 were obtained from ATCC. The HCC cell line SMMC-7721 and the normal human hepatocyte cell line L-02 were purchased from the Chinese Academy of Sciences (Shanghai, China). Hep3B and HepG2 cells were cultured in MEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, and 100 μg/ml non-essential amino acids. SMMC-7721 cells were grown in DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin. L-02 and human umbilical vein endothelial cells (HUVECs) were maintained in RPMI-1640 containing 10% FBS and 100 U/ml penicillin/ streptomycin. miR-451a mimics and miR-451a mimic-Cy3 were purchased from Ribobio Co.