Sirt1 shrna

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).

Recombinant lentiviral vectors (Ad-SIRT1, SIRT1-shRNA, and LDHA-shRNA) were purchased from GeneChem (Shanghai, China). According to user instructions, NP cell transfection is carried out. Through preliminary experiments, the NP infection coefficient (MOI) of this batch of virus is about 15. We add the lentiviral vector when the NP cells reach 30-50% confluence. Observing after 12 hours, if the cells are in good condition, we replace the medium. After three days, we observe the fluorescence to judge the transfection efficiency and replace it with a complete medium containing 2 μg/ml puromycin. We continue to culture for 3-5 days. After the transfection efficiency reaches 90% or more, we use WB or PCR to judge whether the transfection is successful and then use it for subsequent experiments.