Gv362 vector

The GV362 vector and SIRT4 construct were obtained from GeneChem (Shanghai, China), along with the vector element sequence of CMV–MCS–3FLAG–IRES–EGFP–SV40–Neomycin. For the SIRT4 construct, gene-specific primers for SIRT4 were designed: forward primer: 5′-TACCGGACTCAGATCTCGAGCGCCACCATGAAGATGAGCTTTGCGTTGAC-3′; reverse primer: 5′-TCCTTGTAGTCCATGGATCCGCATGGGTCTATCAAAGGCAGC-3′. RT-PCR was performed using a human RNA library as a template. The SIRT4 cDNA was then ligated into the GV362 vector between the XhoI and BamHI restriction sites. The purified fragments were cloned into the vector GV362/SIRT4 and sequenced. Transfection was performed when the seeded cells reached 70–90% confluence. Lipofectamine^TM 3000 was diluted with DMEM. The reagent was mixed well in Opti-MEMTM medium (2 tubes). A master mix of DNA was prepared by diluting DNA in Opti-MEMTM Medium, then adding P3000TM reagent and mixing well. Diluted DNA was added to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio), and incubated for 10–15 min at room temperature. DNA–lipid complexes were added to the cells, and were incubated for two to four days at 37 °C.

shRNA sequences including shNPRA-1: 5’- GCATTCTGATTGTCTCCTTCT-3’, shNPRA-2: 5’-GGGTTGTACTGAACTACAATG-3’ and a nonsense sequence shCTL: 5’-GTTCTCCGAACGTGTCACGT-3’ packaged in lentivirus vectors were designed and synthesized by GenePharma (Shanghai, China). The HIF-1α overexpression plasmid was synthesized into the GV362 vector (Genechem, China). Transfections were performed using lipofectamie 2000 (Invitrogen, USA) following the manufacturer’s instructions.