Genetic Manipulation of HEK293 and HUVEC Cells

HEK 293 cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and transfected with Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). HUVEC cell were cultured in EGM complete medium (Lonza, Allendale, NJ, USA) supplemented with 8% FCS in gelatin-coated dishes and transfected using lentivirus. DNA expressing a short-hairpin RNA (shRNA) designed against human SerRS (5′-GGCATAGGGACCCATCATTGA-3′), GlyRS (5′-GCATGGAGTATCTCACAAAGT-3′), SIRT1 (5′-GAAGTTGACCTCCTCATTGTT-3′) (Guarani et al., 2011 (link)), or SIRT2 (5′-GGACAACAGAGAGGGAGAAAC-3′) gene was inserted into the pLentiLox-hH1 plasmid, modified from the pLentiLox 3.7 plasmid to contain a H1 promoter (between Xba I and Xho I sites) to drive the shRNA expression. To compensate for the loss of endogenous SerRS expression, the coding region for GFP in the pLentiLox-hH1 plasmid was replaced with NLS-deleted or WT (as control) SerRS coding sequences. All designed shRNAs target sequences within the open reading frame except for the SerRS shRNA, which targets the 3′ untranslated region in ordered to selectively knockdown the endogenous gene but not the exogenous genes. The recombinant lentiviruses were produced in packaging 293 cells by cotransfecting the pLentiLox-hH1 plasmid with two helper packaging plasmids Δ8.9 and VSVG and subsequently concentrated by centrifugation at 50,000×g for 3 hr.