Si sirt1

1° and 2° MEFs were transfected prior to reprogramming as previously described [8 (link)]. Briefly, the cells were transfected with si-Sirt1, si-Ku70 and si-Wrn at a concentration of 100 nM (Santa Cruz Biotechnology, Texas, USA). Scrambled siRNA was used as control. Precursor of miR-135a and random sequence precursors (control) at a concentration of 50 nM (Thermo Fisher Scientific). miRCURY LNA inhibitor of miR-135a and random sequence inhibitors (control) at a concentration of 50 nM (Exiqon, Vedbaek, Denmark) were also transfected into the cells. RSV (Sigma-Aldrich) was treated at a concentration of 1 μM.

Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (VA, USA, cat. No. PCS-100-010). Cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37° C with 5% CO₂ in a humidified atmosphere. Replicative senescence of HUVECs was induced by serial passage of HUVECs [52 (link), 53 (link)]. Briefly, cells were harvested and serial passage were performed in the interval of 5-9 days (seed density: 5000 cells/cm²). The passage of cells in control group (“young cell”) were selected when the less than 10% of the population expressed senescence-associated β-galactosidase (SA-β-gal). In contrast, senescence cells (senescence group) were selected under the following conditions [53 (link)]: 1) cells did not increase in numbers during 4 weeks; 2) cells exhibited altered morphology; 3) the positive rate of SA-β-gal staining is more than 70% of population.
Full-length lncRNA Sirt1-AS was subcloned into pcDNA vector (Ke Lei Biological Technology Co., Ltd). Cells were transfected with shSirt1-AS vector (Genechem Shanghai, China), empty pcDNA vector, empty pBABE vector, pcDNA vector (lncRNA Sirt1-AS), pBABE vector (si-Sirt1) and pcDNA vector plus pcDNA-FOXO3a, or with si-Sirt1 (Santa Cruz Biotechnology, Inc.) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).