Lenti sirt1

Lentiviruses were used to generate cells with SIRT1 overexpression or downregulation. Based on the protein expression levels of SIRT1 in several CRC cell lines (Additional file 1: Figure S1A), HCT116 cells were stably transfected with an empty vector (Con077) or SIRT1-shRNA (GeneChem, Shanghai, China). SW480 cells were stably transfected with an empty vector (Con195) or lenti-SIRT1 (GeneChem, Shanghai, China). The transfected cells were then screened with puromycin (4 μg/mL) for several passages. SIRT1 promoter luciferase reporter plasmids (truncated SIRT1 promoter sequences and mutated 100 bp promoter sequences inserted into the GV354 vector, GeneChem, Shanghai, China) as well as EGR1, Sp1 and USF2 overexpression plasmids (constructed using GV230 and GV141 vectors, GeneChem, Shanghai, China) were transiently transfected into HEK-293T cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Lentiviral vectors with the complete coding sequence of SIRT1 (lenti-SIRT1) and NLS-mutant SIRT1 (lenti-SIRT1^NLSmt) and a control empty vector (Con136) were purchased from GeneChem (GeneChem, Shanghai, China). The sites of two NLSs were mutated according to a study conducted by Tanno et al. [17 (link)]. The exogenous proteins of SIRT1 and SIRT1^NLSmt were tagged with green fluorescence protein (GFP). The IGROV1 cells were transfected with Con136, lenti-SIRT1, and lenti-SIRT1^NLSmt and selected with 6 μg/ml puromycin for 48 h. The mRNA and protein expression levels and subcellular location of SIRT1 were confirmed by real-time PCR and Western blot analyses.